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ProtoArray Applications Guide

1. BioEase V5 Control Protein biotinylated V5 tagged control protein A positive control for detection with the Anti V5 Alexa Fluor 647 Antibody and the strepavidin labeled detection reagent Also used as an optional normalization control for immune response serum profiling when anti V5 antibody is added to the detection reagent Human IgA Protein Gradient A positive control for immune response serum profiling of IgA antibodies Interacts with Alexa Fluor 647 anti human IgA Anti Human IgA Antibody Gradient goat anti human IgA A positive control for the immune response serum profiling application Interacts with serum IgA antibodies which are then bound by Alexa Fluor 647 anti human IgA Human IgG Protein Gradient A positive control for the immune response serum profiling application Interacts with Alexa Fluor 647 goat anti human IgG Anti Human IgG Antibody Gradient goat anti human IgG A positive control for the immune response serum profiling application Interacts with serum IgG antibodies which are then bound by Alexa Fluor 647 goat anti human IgG Mdm2 Serves as a control substrate for ubiquitin ligase profiling Yeast calmodulin Cmd1p or human calmodulin CALM2 A positive control for protein protein interaction application and interacts with the Array Control Protein Refer to the lot specific GAL file for the specific identity of the protein GST Protein Grad
2. The raised edges of the LifterSlip should face the surface of the array shown inverted on figure 5 below If air bubbles are observed under the LifterSlip gently raise the LifterSlip and slowly lower it again Raised Pa edges ec 5 Incubate for 90 minutes at 4 C keeping the 4 well tray flat with the array facing up no shaking Add 5 mL cold Washing Buffer and remove the LifterSlip with forceps taking care not to scratch the array surface with the LifterSlip or forceps Wash 5 minutes with gentle agitation Remove Washing Buffer by aspiration see Step 5 of Blocking Step for details Continued on next page Protein Protein Interaction Probing Procedure Continued Probing the Array continued Drying the Array Protocol continued from the previous page 6 7 10 11 12 13 14 15 16 17 Repeat wash steps 4 more times Add 5 mL of primary antibody or Alexa Fluor 647 conjugate see Preparing Antibody Streptavidin Solution Note Always add diluted antibody at the numbered end of the 4 well tray allowing the liquid to flow across the array surface Avoid direct contact with the array and if at all possible avoid applying the antibody solution directly onto the array Incubate for 90 minutes at 4 C with gentle circular shaking 50 rpm Remove primary antibody by aspiration see Blocking Step Wash with 5 mL fresh Washing Buffer for 5 minutes with ge
3. Wash with 5 mL Washing Buffer for 5 minutes using gentle agitation 50 rpm Remove Washing Buffer by aspiration see Blocking Step Repeat wash four more times 10 Proceed to Drying the Array Continued on next page Antibody Specificity Profiling Application Probing Procedure Continued Drying the Array 1 Remove the array from the 4 well tray by inserting the tip of the forceps into the indented numeral and gently prying the edges of the slide upward see figure below Pick up the slide with a gloved hand taking care only to touch the slide only by its edges Tap the slide on its side to remove excess fluid but avoid drying of the array Place on a flat surface or benchtop Place the array in a slide holder or a sterile 50 mL conical tube if you do not have a slide holder Ensure the array is properly placed and is secure in the holder to prevent any damage to the array during centrifugation Briefly dip the slide holder containing the arrays into room temperature distilled water three times to remove salts If you are not using a slide holder dip the array into a 50 mL conical tube filled with room temperature distilled water three times Centrifuge the array in the slide holder or 50 mL conical tube at 200 x g for 1 minute in a centrifuge equipped with a plate rotor if you are using the slide holder at room temperature Verify the array is completely dry After slides have been probed and dried they can be s
4. 3 Using a sterile pipette add 5 mL SMI Assay Buffer into each chamber Avoid pipetting buffer directly onto the array surface 4 Incubate the tray for 1 hour at 4 C on a shaker set at 50 rpm circular shaking Use a shaker that keeps the arrays in one plane during rotation Rocking shakers are not to be used because of increased risk of cross well contamination 5 After incubation aspirate SMI Assay Buffer by vacuum or with a pipette Position the tip of the aspirator or pipette into the indented numeral and aspirate the buffer from each well see figure 2 Tilt the tray so that any remaining buffer accumulates at the end of the tray with the indented numeral Aspirate the accumulated buffer Important Do not position the tip or aspirate from the microarray surface as this can cause scratches Immediately proceed to adding the next solution to prevent any part of the array surface from drying which may produce high or uneven background 6 Proceed immediately to Probing the Array Continued on next page Small Molecule Interaction Probing Procedure Continued Probing the Arra 1 with Alexa Fluor Labeled Probe Remove the slide from the 4 well tray using forceps see figure 3 below and tap on a paper towel to remove excess fluid Place on a flat surface Insert the tip of the forceps into the indented numeral and gently pry the edges of the slide upward Pick up the slide with a gloved hand taking care only to touc
5. 6 Proceed immediately to Probing the Array Continued on next page 102 Immune Response Biomarker Profiling Probing Procedure Continued Probing the Array 10 11 12 13 14 Add 5 mL Washing Buffer at the indented numeral end of the 4 chamber incubation tray without touching the array surface Incubate the tray for 5 minutes at 4 C on a shaker set at 50 rpm circular shaking Aspirate the buffer using vacuum or pipette as described on the previous page Step 5 Add 5 mL serum or plasma diluted 1 500 in Washing Buffer at the indented numeral end of the 4 chamber incubation tray without touching the array surface Allow the sample to flow across the array surface Avoid pipetting sample directly onto the array surface Incubate the tray for 90 minutes at 4 C on a shaker set at 50 rpm circular shaking Aspirate the sample using vacuum or pipette as described on the previous page Step 5 Wash each array with 5 mL Washing Buffer with gentle shaking on a shaker set at 50 rpm for 5 minutes at room temperature Aspirate the Washing Buffer as described on the previous page Step 5 Repeat Step 6 four more times using fresh Washing Buffer each time to obtain a total of 5 wash steps During the wash steps mix 2 5 uL Alexa Fluor 647 goat anti human IgG antibody with 5 mL Washing Buffer per array to obtain a final antibody concentration of 1 ug mL Store on ice until use Optional add Alexa Fluor 647 label
6. General guidelines for using the ProtoArray Microarrays are described below Review this section before starting the probing procedure Choose the appropriate probing protocol based on the application that you wish to perform Application Page no Protein Protein Interaction PPI 12 Kinase Substrate Identification KSI 35 Small Molecule Identification SMI Fluorescent 56 3H Labeled Small Molecule Identification SMI Radioactive 71 Ubiquitin Ligase Profiling 84 Immune Response Biomarker Profiling IRBP 97 Antibody Specificity Profiling ASP 109 Since proteins are sensitive to various environmental factors each array is produced in an environment controlled facility to ensure protein integrity and maintain consistency To obtain the best results and avoid any damage to the array or array proteins always handle the ProtoArray Microarray with care using the following guidelines e ProtoArray Microarrays can only be used once Do not re use the array or re probe the same array with another probe e Always wear clean gloves while handling the microarray e Do not touch the surface of the array Damage to the array surface can result in uneven or high background e Maintain the array and reagents at 2 8 C during the experiment unless otherwise specified e Prevent condensation on the array by equilibrating the mailer containing the array at 4 C for at least 15 minutes prior to removing the array Immerse the array immedi
7. This file contains a description of control spots on the array Slide Information File LotNumber slide txt This file contains a listing of all barcodes associated with a specific lot of arrays Note The file size for some files such as the Protein Sequence File may be larger than 1MB TM Continued on next page 127 Data Acquisition and Analysis Continued Data Acquisition Data Analysis Using ProtoArray Prospector 128 Data acquisition software is used to obtain pixel intensity information for each spot feature on the array Information on additional parameters may be recorded depending on the type of software used for data acquisition 1 Start the microarray data acquisition software on the computer and open the saved image tiff from Step 8 page 125 2 To acquire data from ProtoArray experiments e For GenePix Pro Software download the GAL files from ProtoArray Central for protein arrays which defines the array grid required by the microarray data acquisition software e For other microarray data acquisition software use data from the GAL files from ProtoArray Central for protein arrays to generate files that are compatible with your microarray data acquisition software to define the array grid Scroll through the image to ensure that the grid is in the proper location for each subarray Adjust the subarray grid if needed Utilize the automatic spot finding function of the image acquisition
8. 49 Image Acquisition and Processing Continued ProtoArray Prospector Results The Next Step 50 After data analysis ProtoArray Prospector presents a summary of the analyzed data in a table format see ProtoArray Prospector manual for details The proteins that score as positive in the experiment are proteins that satisfy the basic program options We recommend reproducing the results using ProtoArray Technology or other methods as described below After identifying potential kinase substrates on the ProtoArray Human Microarray you may reproduce the result using The ProtoArray Technology with additional arrays to ensure e Reproducibility Probe the human array using a similar or a different kinase concentration to address reproducibility e Specificity Probe a human array with different kinase to identify substrates specific to your protein kinase of interest OR A solution assay as described briefly below To verify substrate phosphorylation in solution perform solution assays in the presence of radiolabeled ATP using the purified protein kinase and potential kinase substrate using the probing conditions described in this manual Be sure to include appropriate positive and negative control reactions Analyze the results using SDS PAGE and autoradiography A true positive signal identified on the array should also produce positive results using the solution assay while a false positive signal identified on the a
9. 647 The fluorescent antibody signals indicate that the array has been properly scanned and are used as reference spots to orient the microarray and help assign spot identities e Anti biotin Ab signal Biotinylated proteins bind to the Anti biotin antibody printed on the microarray e BioEase biotin V5 Control Protein signal The Anti V5 Alexa Fluor 647 Antibody binds to a control protein V5 Control containing an N terminal V5 tag printed on the microarray The signals indicate that the antibody is functional and probing is performed properly The signal is also used to check the background The Streptavidin Alexa Fluor 647 conjugate also binds to the biotinylated V5 tagged control protein V5 Control printed on the microarray The signals indicate that the probing is performed properly e Biotin Ab signal A biotinylated anti mouse antibody is printed on the microarray The Streptavidin Alexa Fluor 647 conjugate and the mouse Anti V5 Alexa Fluor 647 antibody binds to the biotinylated anti mouse antibody 30 Continued on next page Expected Results for PPI Continued Human Array Results obtained after probing the ProtoArray Human Protein Microarray v5 0 Probing Results with the Array Control Protein i e BioEase V5 tagged biotinylated calmodulin kinase are shown below Human Array probed with 50 pg mL Array Control Protein and Streptavidin Alexa Fluor 647 Conjugate Human Array probed with 5 pg mL Array C
10. Probing the Array 1 Pipet5 mL of Assay Buffer page 88 on top of the barcode without touching with Ubiquitin the array surface Ligase Mixture 2 Incubate 3 minutes at 4 C with gentle shaking 50 rpm 3 Remove the array from the 4 well tray by inserting the tip of the forceps into the indented numeral and gently prying the edges of the slide upward see figure 3 Pick up the slide with a gloved hand taking care only to touch the slide only by its edges Tap the slide on its side to remove excess fluid but avoid drying of the array Place on a flat surface or benchtop 4 Pipet 100 uL of ubiquitin ligase probe mixture onto the array dropwise Make sure the pipette tip does not touch the surface of the array Gently rock the slide about 15 30 seconds to spread the solution and then using forceps gently overlay the LifterSlip white rails on the slip facing the array Be careful to not trap bubbles during this step If bubbles are observed lift the slip with forceps and slowly lower the slip again 5 Incubate for 90 minutes at 30 C in a humidified chamber or a sealed plastic bag with a wet paper towel keeping the 4 well tray on a flat surface with the arrays facing up no shaking Continued on next page 90 Ubiquitin Ligase Probing Procedure Continued Probing the Array 6 Add5mL Assay Buffer to incubation tray and incubate without agitation with Ubiquitin After about a minute or so the LifterSlip should float off
11. Thermo Scientific Cat no 25x601 2 4789 e Microarray slide holder and centrifuge equipped with a plate holder Optional The microarray is placed in an incubation tray during the blocking and washing steps To obtain the best results all incubations of the ProtoArray with various solutions are performed in a 4 chamber covered incubation tray Greiner Cat no 96077307 LifterSlip coverslips Thermo Scientific Cat no 25x601 2 4789 hold a small reagent volume to minimize the amount of valuable probe used and prevent evaporation of reagents If you are using any other coverslip be sure the coverslip is able to completely cover the printed area 20 mm x 60 mm of the glass slide and the coverslip is made of non protein binding material Untreated glass coverslips are not recommended Continued on next page Ubiquitin Ligase Probing Procedure Continued Using Your Own Buffers Preparing Ubiquitin Ligase Mixture Preparing Streptavidin Solution Preparing 0 5 SDS Follow the guidelines listed below for buffer preparation to obtain the best results with microarrays The buffer recipes are listed below e Always use ultra pure water to prepare reagents and buffers e You may use non ionic detergents and reducing agents during probing to minimize non specific interactions e If the protein interaction requires certain co factors be sure to include the co factors in the probing buffer during probing e Prepare
12. by implication by estoppel or otherwise In particular the purchase of this product does not include or carry any right or license to use develop or otherwise exploit this product commercially This product is sold pursuant to authorization from Incyte Corporation and Incyte Corporation reserves all other rights under these patents For information on purchasing a license for purposes other than research and development please contact Incyte Corporation Corporate Licensing Route 141 and Henry Clay Boulevard Wilmington DE 19880 Phone 302 498 6825 Fax 302 498 2707 The purchase of this product conveys to the buyer whether employed in academia government not for profit entity or a for profit entity the limited non exclusive non transferable right without the right to resell repackage or further sublicense to use this product solely for its own research and development purposes No other license is granted to the buyer whether expressly by implication by estoppel or otherwise In particular the purchase of this product does not include or carry any right or license to use develop or otherwise exploit this product commercially such as the generation of commercial databases or the provision of clinical diagnostics or for testing the binding or activity of a small molecule or antibody against a protein for the purposes of identifying potential pharmacological and or therapeutic action This product is sold pursuant to authorization
13. protein kinases available from Invitrogen it is very easy to order the clone or purified protein corresponding to the protein identified on the array and validate the interaction Visit www invitrogen com clones to access our clone collections Each Ultimate ORF Clone is full insert sequenced and guaranteed to match the corresponding GenBank amino acid sequence Contact Technical Support page 137 to order the purified protein kinases printed on the array or to request information about custom production of additional proteins present on the array Technical Support Web Resources Visit the Invitrogen Web site at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 650
14. with Biotin Labeled Probe 64 1 10 11 12 Remove the slide from the 4 well tray using forceps by inserting the tip of the forceps into the indented numeral and gently prying the edges of the slide upward see figure 3 below Pick up the slide with a gloved hand taking care only to touch the slide only by its edges Tap the slide on its side to remove excess fluid but avoid drying of the array Place on a flat surface or benchtop Pipet 120 uL of the small molecule diluted in SMI Assay Buffer page 60 on top of the array without touching the array surface with the pipette tip dropwise Gently rock the slide about 15 30 seconds for solution to spread Using forceps carefully lay the LifterSlip coverslip on the array to cover the printed area without trapping any air bubbles If bubbles are observed gently lift the LifterSlip and slowly lower the slip again Replace slide in the 4 well tray and cover with lid Incubate 90 minutes at 4 C Add 5 mL SMI Assay Buffer incubate without agitation and remove LifterSlip After about a minute or so the LifterSlip should float off of the ProtoArray Human Protein Microarray Once this occurs use the forceps to carefully remove the LifterSlip Discard the slip Alternatively remove the array and LifterSlip from the well and tilt the slide to allow the LifterSlip slip off the surface Replace the array back into the incubation tray to Wash with 5 mL SMI Ass
15. Assay Buffer Optional If you are a first time user of the ProtoArray Human Protein Microarray perform a control probing using a ProtoArray Control Microarray to verify probing and detection protocols 3 Dry the microarray 65 Scan slide with fluorescence microarray scanner 66 Download the protein array lot specific information the 66 GAL file from ProtoArray Central Portal to acquire and analyze the data using ProtoArray Prospector to identify small molecule interactions Continued on next page 56 Guidelines for Probing the ProtoArray Microarray Human Protein The recommended small molecule probe concentration for probing the Microarray ProtoArray Human Protein Microarray is at least 2 5 uM Probing Options A number of options are available for probing the ProtoArray Human Protein Microarray with your own buffers and detection reagents as described below Review the information below before proceeding with the probing procedure Probing options can be performed individually or in tandem and include e Probing with your small molecule probe to detect novel interactions e Probing with only the detection reagent negative control The negative control allows you to determine signals specific to your probe e Probing with different probe concentrations to determine the optimal amount of probe for your assay Start with an initial probe concentration If the initial signal is strong with
16. Buffer final concentration 1X PBS 1X Synthetic Block 0 1 Tween 20 1 Prepare 60 mL of buffer for each microarray For 1 000 mL Washing Buffer prepare fresh reagents as follows 10X PBS pH 7 4 100 mL 10X Synthetic Block 100 mL 10 Tween 20 01 mL Deionized water to 1 000 mL 2 Mix well and store on ice until use ProtoArray Human Protein Microarray To probe the microarray you need 120 uL of your protein probe containing a suitable tag The recommended protein probe concentration range for probing the ProtoArray Human Protein Microarray is 100 nM 10 uM for biotinylated proteins and 10 nM 1 uM for V5 tagged proteins Dilute the probe to the recommended starting concentration in Washing Buffer Mix well do not vortex and store on ice until use ProtoArray Control Protein Microarray For V5 tag detection Mix 12 uL Array Control Protein included with the array to a final volume of 120 uL with Washing Buffer Mix well do not vortex and store on ice until use For biotin detection Mix 12 uL Array Control Protein included with the array to a final volume of 120 uL with Washing Buffer Mix well do not vortex and store on ice until use Continued on next page Protein Protein Interaction Probing Procedure Continued Preparing Antibody Streptavidin Solution ProtoArray Human Protein Microarray The protein probe is detected using a primary or secondary fluorescent conjugate Any primary antibody specific
17. For detailed instructions on scanning the microarray refer to Image Acquisition and Processing for Radioactive Assays page 129 1 Develop the phosphor screen according to the manufacturer s recommendations 2 Scanthe phosphor screen on a phosphorimager to generate a 16 bit TIFF image file 3 Process the image using ProtoArray Prospector Imager 4 Save the adjusted microarray image For detailed instructions on Data Acquisition and Analysis refer to page 131 1 Acquire an image tiff from the phosphor screen 2 Usethe barcode information on the array to download the GAL file from ProtoArray Central as described on page 132 3 Usethe GAL file and ProtoArray Prospector to acquire pixel intensity values for all features on the array and analyze data to determine significant signals Continued on next page 79 Image Acquisition and Processing Continued ProtoArray After data analysis ProtoArray Prospector presents a summary of the analyzed Prospector data in a table format see ProtoArray Prospector manual for details Results The proteins that score as positive in the experiment are proteins that satisfy the basic program options We recommend reproducing the results using ProtoArray Technology or other methods as described below The Next Step After identifying potential small molecule interactions on the ProtoArray Human Microarray you may reproduce the result using The ProtoArray Technology with a
18. Human Protein Microarray specifications are listed below Dimensions 1 inch x 3 inch 25 mm x 75 mm Material Glass slide coated with a thin layer of nitrocellulose The nitrocellulose coated slide is from GenTel BioSciences Inc Thin film nitrocellulose slides are manufactured by Gentel Biosciences Inc using a proprietary surface chemistry owned by Decision Biomarkers Inc Thin film nitrocellulose slides are covered by US Patent 6 861 251 7 297 497 and 7 384 742 Each microarray has a barcode for tracking samples The barcode number is also used to retrieve array specific information from the ProtoArray Central Portal page 126 The ProtoArray Human Protein Microarray specifications are listed below The proteins on the microarray are printed in 48 subarrays that are equally spaced in vertical and horizontal directions For details on the subarray layout and human protein and control spots on the ProtoArray Human Protein Microarray go to the ProtoArray Central Portal at www invitrogen com protoarray Total Subarrays 48 4 columns x 12 rows Subarray Size 4 400 uM x 4 400 uM Subarray Dimensions 22 rows x 22 columns Median Spot Diameter 110 uM Spot Center to Center Spacing 200 uM Distance Between Subarrays 100 uM Replicates per Sample 2 Total Human Proteins on v5 0 Array gt 9 000 Refer to ProtoArray Central Portal for exact number of human proteins printed on the microarray Continued on next
19. Improper washing To obtain the best results perform the recommended washing steps Prepare the Washing Buffer fresh as described on page 113 Array dried during probing Do not allow the array to dry during probing Array not dried properly Dry the array before scanning before scanning High antibody Decrease the antibody concentration or decrease concentration the incubation time Antibody cross reactivity Probe a protein array using only the secondary antibody without the antibody sample to detect cross reactivity with the secondary antibody only Continued on next page 121 Troubleshooting Continued Uneven background Uneven blocking or During the blocking or washing steps ensure the washing array is completely immersed in blocking solution or Washing Buffer and use 5 mL buffer in the each chamber of the incubation tray to cover the array completely with buffer Improper washing To obtain the best results perform the recommended washing steps Prepare the Washing Buffer fresh as described on page 113 Portions of array have dried Do not allow the array to dry during probing Improper array handling Always wear gloves and avoid touching the surface of the array with gloved hands or forceps Take care while inserting or removing the array from the incubation tray to avoid scratching the array surface Antibody sample or Centrifuge the antibody sample or detection detection reagents contain reagents to remove
20. Introduction The table below provides some solutions to possible problems you may encounter when using the ProtoArray Microarray for the KSI application Weak or no signal Kinase of interest is not Check the activity of the kinase after purification with your protein active or is inactivated by using a method of choice Ensure the kinase is kinase the assay buffer active under the conditions used for probing Avoid repeated freezing thawing of your kinase Low specific activity of the Perform probing with higher kinase kinase concentration higher kinase specific activity or increase the incubation time Avoid repeated freezing thawing of your kinase Incorrect scanning or For X ray film develop the film and acquire the imaging image using a standard scanner For phosphor screen acquire the image using a phosphorimager Follow the manufacturer s recommendations on using the scanner or phosphorimager to scan the array correctly Be sure to use a scanner or phosphorimager that provides at least 50 uM resolution and generates 16 bit TIFF image files Incorrect assay conditions Perform incubation of the array at 30 C during the probing procedure Use freshly prepared Kinase Buffer for best results Poor incorporation of Use fresh y P ATP Be sure to check the array radiolabel using a Geiger counter to verify that the radioactive signal is obtained after the probing procedure Kinase ATP mixture not After preparing the kinas
21. Methods Alexa Fluor Detection Important 14 An appropriate detection system is required to perform the protein protein interaction application see below Various options are available for performing the probing procedure see next page for details An experimental workflow for probing the human and control microarray is shown on pages 16 17 Fluorescence detection is used to detect protein protein interactions on ProtoArray Microarrays Fluorescent detection offers high sensitivity low background and signal stability Select the appropriate detection method based upon the nature of your probe Epitope Tag To detect an epitope tag on your protein probe use a labeled antibody specific to the tag The antibody can be directly labeled with a fluorescent dye or detected through a secondary antibody conjugated to a fluorescent dye Biotin Label To detect a biotin label on your protein probe use streptavidin conjugated to a fluorescent dye for signal amplification and increased sensitivity The Alexa Fluor detection system available from Invitrogen page 135 is the recommended fluorescent detection method The Alexa Fluor 647 fluorophore is brighter and more stable than other commercially available dyes such as Cy5 dyes and is more sensitive for detecting interactions on protein arrays We have demonstrated that detection with Alexa Fluor 647 produces approximately 2 fold higher signal background ratios than Cy5 det
22. Probing Procedure Continued Preparing Blocking Buffer Preparing Washing Buffer Blocking Buffer use 5 mL buffer per microarray 50 mM HEPES pH 7 5 200 mM NaCl 0 08 Triton X 100 25 Glycerol 20 mM Reduced glutathione 1X Synthetic Block 1 mM DTT 1 Prepare 100 mL Blocking Buffer fresh as follows 1M HEPES pH 7 5 5mL 5M NaCl 4mL 10 Triton X 100 800 uL 50 Glycerol 50 mL Reduced glutathione 610 mg 10X Synthetic Block 10 mL Deionized water to 100 mL 2 Mix reagents adjust pH to 7 5 with NaOH and add 100 pL of 1 M DTT prior to use 3 Use buffer immediately and store any remaining buffer at 4 C for lt 24 hours Washing Buffer use 60 mL buffer per microarray 1X PBS 0 1 Tween 20 1X Synthetic Block 1 Prepare 1 000 mL Washing Buffer fresh as follows 10X PBS 100 mL 10 Tween 20 10 mL 10X Synthetic Block 100 mL Deionized water to 1 000 mL 2 Mix reagents and cool to 4 C 3 Use buffer immediately Remaining buffer can be stored at 4 C for 24 hours 101 Immune Response Biomarker Profiling Probing Procedure Continued Before Starting e Before starting the probing procedure make sure you have all items on hand especially buffers see page 101 serum or plasma sample diluted in Washing Buffer and incubation tray see page 100 e Make sure the buffers are cold and stored on ice until use Place an incubation tray on ice to chill until use e Review Important Guidelines on page 11 prior
23. X ray film or a phosphor screen for 3 hours Acquire the array image to produce a 16 bit TIFF file The array image can be acquired by scanning the phosphor screen using a phosphorimager or develop the X ray film and scan the X ray film using a scanner Process the microarray images and acquire and analyze data using ProtoArray Prospector recommended Do not use Y P ATP for the assay use y P ATP as the use of y P ATP supports increased signal resolution during data acquisition While y P ATP can be used for the assay data quantitation with y P ATP is not supported Incubation chambers are not suitable for use in the probing portion of the KSI application A container that seals tightly is required to prevent any leakage of radioactive material during the washing steps Do not use cold ATP for the kinase probing steps If your kinase is stored in a buffer containing ATP make sure the final concentration of cold ATP is less than 100 nM during the kinase probing step Avoid adding more than 10 v v of the kinase sample to 120 uL of Kinase Buffer Addition of more than 10 of the kinase to the Kinase Buffer can decrease assay performance Continued on next page 41 Kinase Substrate Identification Probing Procedure Continued Materials Needed Coverslips Using Your Own Buffers 42 ProtoArray Human or Control Protein Microarray v5 0 Note You need to purchase an additional ProtoArray Human or Control Pr
24. a list of scanners to use with ProtoArray Microarrays see page 124 Data acquisition software We recommended GenePix Pro v6 or later Molecular Devices Corporation or ScanArray Acquisition Software PerkinElmer Inc as microarray data acquisition software for analysis of images For detailed instructions on scanning the microarray refer to Scanning Arrays Using a Fluorescence Scanner page 123 Insert array into the fluorescence microarray scanner Adjust scanner settings Preview the microarray and adjust settings if needed Scan the microarray Save image data Q gn moo qur Export and analyze results For detailed instructions on Data Acquisition and Analysis refer to page 126 1 To acquire data from the scanned image use the barcode on the array to download the GAL file from ProtoArray Central as described on page 126 2 Use the GAL file and suitable microarray data acquisition software to acquire pixel intensity values for all features on the array 3 Analyze data with ProtoArray Prospector using the guidelines on page 128 to determine significant signals with the controls and your protein probe Note Set the Application in ProtoArray Prospector to Immune Response Profiling for serum samples or to Immune Response Profiling with Plasma for plasma samples After data analysis ProtoArray Prospector presents a summary of the analyzed data in a table format see ProtoArray Prospector manual for de
25. are available from Invitrogen for labeling of your small molecule of interest For more information about these products refer to our website www invitrogen com or call Technical Support page 135 Biotin Tag You may use any method to biotinylate your small molecule of interest To label your small molecule probe with a biotin tag your small molecule of interest must contain the appropriate functional group for labeling e When performing fluorescence detection it is important to avoid exposing the array to light after probing with a fluorescent detection reagent e If performing direct labeling always verify that labeling does not affect the binding affinity of the antibody e Although Alexa Fluor 555 or Cy3 dyes can be used for detection using them may result in higher background signals Continued on next page Small Molecule Interaction Probing Procedure Introduction After preparing the small molecule probe and verifying the presence of the tag or label probe the ProtoArray Human Protein Microarray using your small molecule probe Instructions are included in this section to probe the ProtoArray Human Protein Microarray using buffer recipes provided in this manual see page 60 for buffer recipes Experimental 1 Block the ProtoArray Human Protein Microarray Outline 2 Probe with your tagged small molecule probe 3 Perform detection using an appropriate detection system 4 Dry the array for scanning
26. array surface Apply the probe solution and LifterSlip or equivalent coverslip to the array as described in the manual To avoid drying of the array surface make sure the coverslip covers the printed area of the array and adjust the coverslip if needed Probe or detection reagents Centrifuge the probe or detection reagents to contain precipitates remove precipitates prior to probing the array Continued on next page Immune Response Biomarker Profiling IRBP Application Experimental Overview Experimental The experimental outline for performing IRBP application using the ProtoArray Outline Human Protein Microarray with serum samples is shown below Step Action Page no 1 Block the ProtoArray Human Protein Microarray 102 2 Probe the ProtoArray Human Protein Microarray with the 103 diluted serum sample and perform detection using a suitable detection system Optional If you are a first time user of the ProtoArray Human Protein Microarray perform a control probing using a ProtoArray Control Microarray to verify probing and detection protocols 3 Dry the microarray 104 4 Scan the microarray using a suitable microarray scanner and 105 save an image of the array 5 Download the protein array lot specific information the 105 GAL file from ProtoArray Central Portal to acquire and analyze the data using ProtoArray Prospector to identify significant protein protein interactions Exper
27. buffers fresh prior to use Freshly prepared blocking buffer is best for blocking slides e Use the recipes described below to prepare your own buffers Recommended buffers are listed below for blocking and washing the arrays You can perform array probing using the recommended buffers and then based on your initial results optimize the buffer formulation Ubiquitin Ligase Mixture To probe the microarray you need 100 pL of your ubiquitin ligase mixture with labeled ubiquitin for each array We recommend the following concentrations as a starting point 1 Add 0 1 mg mL Biotin Ubiquitin 2 Add ubiquitin conjugating enzymes e 100 nM ubiquitin activating enzyme E1 e 10 100 nM ubiquitin conjugating enzyme E2 e 10 250 nM ubiquitin ligase enzyme E3 3 Add 1X Energy Regenerating Solution Boston Biochem Cat no B 10 or 20 mM ATP in Assay Buffer 4 Mix well do not vortex and store on ice until use Prepare 5 mL of Streptavidin Alexa Fluor 647 Conjugate in Assay Buffer at 1 pg mL for each array to be probed Prepare 15 mL of 0 5 SDS for each microarray For 200 mL of 0 5 SDS prepare the following reagents fresh from 10 SDS as follows 10 SDS 10 mL Ultrapure water 190 mL Total Volume 200 mL Mix well and store at room temperature until use Continued on next page 87 Ubiquitin Ligase Probing Procedure Continued Preparing Blocking Buffer Preparing Assay Buffer 88 Blocking Buffer final concentration 5
28. included in this section for probing the ProtoArray Human Protein Microarray for IRBP using your diluted serum or plasma sample Follow the guidelines provided in this section Use the probing procedure from this section as a starting protocol Based on your initial results you may need to optimize the probing protocol by varying serum or plasma concentrations Block the ProtoArray Human Protein Microarray with Blocking Buffer Probe the array with diluted 1 500 human serum or plasma Perform detection using Alexa Fluor 647 goat anti human IgG Dry the array for scanning Ol a O Scan the array with a fluorescence microarray scanner to obtain an array image 6 Download the protein array lot specific information from ProtoArray Central portal and acquire the image data using microarray data acquisition software 7 Analyze results using ProtoArray Prospector data analysis software available from www invitrogen com protoarray e ProtoArray Human Protein Microarray v5 0 e Human serum or plasma sample dilute the sample 1 500 in Washing Buffer store on ice until use e Blocking Buffer and Washing Buffer see page 101 for recipes e 10X Synthetic Block see page 135 e Alexa Fluor 647 Goat Anti Human IgG Invitrogen Cat no A21445 e Clean covered 4 chamber incubation tray Greiner Cat no 96077307 chilled on ice e Forceps and deionized water e Shaker capable of circular shaking at 50 rpm place the shak
29. low background confirm the initial results with a second array using the same experimental conditions If the initial results indicate weak signal or an unacceptable signal to noise ratio probe a second array with a different probe concentration 57 Preparing the Small Molecule Probe Introduction Small Molecule Tags Generating Tagged Small Molecule Probe Important 58 Before using the ProtoArray Human Protein Microarray your small molecule of interest must contain a suitable tag to probe the microarray The amount and quality of your small molecule required for probing are described in this section The small molecule of interest can be tagged using a reactive Alexa Fluor dye or a biotin label Using amine or sulhydryl reactive Alexa Fluor dyes small molecules with the appropriate functional group can be directly labeled for use as a probe We recommend the use of reactive Alexa Fluor 647 to obtain the best results The extremely high affinity of the biotin streptavidin interaction makes biotin protein conjugation an attractive method for probe labeling The biotinylated small molecule probe is detected using a streptavidin detection system Alexa Fluor Tag To label your small molecule probe with an Alexa Fluor tag your small molecule of interest must be contain the appropriate functional group which will allow labeling with a reactive Alexa Fluor dye A variety of reactive Alexa Fluor 647 dyes
30. of 10 50 nCi uL A number of options are available for probing the human microarray with a small molecule of interest using your own buffers and detection reagents as described below Review the information below before proceeding with the probing procedure Probing options can be performed individually or in tandem and include e Probing with your tritiated small molecule of interest to identify potential substrates e Probing with H estradiol positional mapping reagent The result from the positional mapping reagent can serve as a positive control to help determine signals specific to your probe e Probing with different probe concentrations to determine the optimal amount of probe for your assay Start with an initial probe concentration If the initial signal is strong with low background confirm the initial results with a second array using the same experimental conditions If the initial results indicate weak signal or an unacceptable signal to noise ratio probe a second array with a different probe concentration Continued on next page Tritium Radiolabeled Small Molecule Interaction Probing Procedure Introduction Experimental Outline Materials Needed Coverslips After preparing the small molecule probe and verifying the presence of the label probe the ProtoArray Human Protein Microarray using your small molecule probe Instructions are included in this section to probe the ProtoArray Human Protein
31. of the ProtoArray Ligase Mixture Human Protein Microarray Once this occurs use the forceps to carefully continued remove the LifterSlip Discard the slip Alternatively remove the array and LifterSlip from the well and tilt the slide to allow the LifterSlip to slip off the surface Replace the array back into the incubation tray Remove Assay buffer by aspiration see Figure 2 Wash with 5 mL 0 5 SDS with gentle agitation for 5 minutes Aspirate 0 5 SDS see Figure 2 Repeat wash step two more times 9 Wash with 5 mL Assay buffer with gentle agitation for 5 minutes Aspirate Assay Buffer see Figure 2 Repeat wash step one more time 10 Add 5 mL streptavidin Alexa Fluor 647 diluted in Assay Buffer at 1 ug mL Add streptavidin Alexa Fluor 647 at the indented numeral end of the 4 well tray and allow the liquid to flow across the slide surface To prevent local variations in fluorescence intensity and background avoid direct contact with the slide 11 Incubate for 45 minutes at at 4 C with gentle shaking 50 rpm 12 Remove streptavidin Alexa Fluor 647 solution by aspiration 13 Wash with 5 mL Assay Buffer with gentle agitation for 5 minutes Aspirate Assay Buffer Repeat wash step four more times 14 Remove the array from the 4 well tray using forceps 15 Proceed to Drying the Array Drying the Array 1 Remove the array from the 4 well tray using forceps by inserting the tip of forceps into the indented
32. orient the results obtained from the GAL file and ProtoArray Prospector with the array image position the microarray image such that the barcode is at the bottom of the image In this orientation the top left corner of the microarray image is Block 1 Continued on next page 81 Troubleshooting Introduction The table below provides some solutions to possible problems you may encounter when using the ProtoArray Microarray for the SMI Radioactive applications Weak or no signal Low specific activity of the Perform probing with higher small molecule with your small small molecule concentration higher small molecule specific molecule activity or increase the incubation time Incorrect scanning or For phosphor screen acquire the image using a imaging phosphorimager Follow the manufacturer s recommendations on using the scanner or phosphorimager to scan the array correctly Be sure to use a scanner or phosphorimager that provides at least 50 uM resolution and generates 16 bit TIFF image files Incorrect assay conditions Perform incubation of the array at 30 C during the probing procedure Use freshly prepared Tritium SMI Assay Buffer for best results Poor incorporation of Be sure to check the array using a Geiger counter radiolabel to verify that the radioactive signal is obtained after the probing procedure Small molecule specific Use another small molecule substrates are not present on the array Poor spot resol
33. page ProtoArray Human Protein Microarray Continued Array Content The majority of the human protein collection is derived from the human Ultimate ORF open reading frame Clone Collection available from Invitrogen see orf invitrogen com for more information Each Ultimate ORF Clone is full insert sequenced and is guaranteed to match the corresponding GenBank amino acid sequence Some of the human proteins printed on the array represent the human protein kinase collection derived from full insert sequenced clones but are not Ultimate ORF Clones Some of the kinases from the kinase collection have been cloned as catalytic domains rather than full length proteins About 313 proteins printed on the array are derived from the purified protein kinase collection available from Invitrogen Approximately 40 additional proteins printed on the array are purified cytokines available from Invitrogen Approximately 28 proteins peptides and nucleic acids that have been demonstrated to be antigens in a variety of autoimmune diseases are also printed on the array New content for ProtoArray v 5 0 arrays was enriched for proteins relevant to disease processes for a total of 26 100 potential drug targets printed on the array For accession number and amino acid sequence for each protein as well as information on peptides and nucleic acids printed on the array download the Protein Content List from www invitrogen com protoarray as described o
34. performing experiments with additional arrays to ensure e Reproducibility Probe protein arrays using a similar or a different probe concentration to observe similar interactions e Specificity Probe protein arrays with the detection reagent used to visualize the interactions and also different proteins containing the tag to identify interactions specific to your protein probe of interest and also identify any non specific interactions e Reciprocal Interactions Determine reciprocal interactions using a purified protein probe see below Other methods for validating protein protein interactions include e Yeast Two Hybrid Systems page 135 e Co immunoprecipitation e Gel shift assay The ProtoArray Human Protein Microarray is ideal for detecting reciprocal protein protein interactions since proteins are purified under native conditions and the microarrays are manufactured under highly controlled conditions to ensure maximum protein function Once you have identified a positive interaction if your original protein probe is present on the ProtoArray Human Protein Microarray v5 0 you can use the identified interacting protein from the array as a probe for probing another human microarray For example perform an initial probing with calmodulin as a probe with a ProtoArray Human Protein Microarray to detect the interacting protein calmodulin kinase Then perform the reciprocal interaction with another human microarray using calmo
35. probability that the control or human protein spot is missing from the entire lot is then calculated The percentage of missing spots is estimated as the average missing probability of all the spots That estimation must indicate that at least 9576 of spots are present Consistent print quality is determined for all sub arrays prior to starting the printing of each array lot Proteins of a particular type or class are distributed randomly across all sub arrays and therefore several spots missing from a single sub array is essentially no different from random spots missing across several sub arrays The control features are functionally qualified by probing with control proteins to detect the appropriate interactions Control Proteins Various proteins and controls are printed on each ProtoArray Human Protein Microarray to verify detection conditions and background See page 9 for details ProtoArray Control Protein Microarray Introduction Important Control Microarray Specifications The ProtoArray Control Protein Microarray contains various controls printed on a nitrocellulose coated glass slide The ProtoArray Control Protein Microarray allows you to validate probing procedures prior to probing the ProtoArray Human Protein Microarray If you are first time user of the ProtoArray Technology we recommend that you probe a ProtoArray Control Protein Microarray prior to probing the ProtoArray Human Microarray If you are usi
36. the end of the tray see figure 1b and aspirate the buffer from each well see figure 2 Tilt the tray so that any remaining buffer accumulates at the base of the well at the numbered end of the tray and aspirate Important Do not position the tip on or aspirate from the microarray surface as this can cause scratches Immediately proceed to adding the next solution to prevent any part of the array surface from drying 7 Proceed immediately to Probing the Array Continued on next page 23 Protein Protein Interaction Probing Procedure Continued Probing the Array 26 Instructions for probing the microarray are described below 1 Remove array from the 4 well tray by inserting the tip of forceps into the indentation at the numbered end of the tray and gently prying the array upward see figure 3 below Pick up array with a gloved hand taking care to only touch the array by its edges Gently dry the back and sides of the array on a paper towel to remove excess buffer Note To ensure that the array surface remains wet do not dry more than 2 arrays at a time before adding the diluted probe and LifterSlip coverslip Pipet 120 uL of the probe in Washing Buffer page 22 on top of array without touching the array surface The liquid should spread over the surface of the array Carefully lower a LifterSlip coverslip over the printed area of the array using forceps as shown below figure 4 below ee a m
37. the microarray is near the tray end containing an indented numeral see figure 1a and 1b 3 Using a sterile pipette add 5 mL Tritium SMI Assay Buffer into each chamber Avoid pipetting buffer directly onto the array surface 4 Incubate the tray for 1 hour at 4 C on a shaker set at 50 rpm circular shaking 5 After incubation remove ProtoArray Protein Microarrays from Tritium SMI Assay Buffer To remove array from the 4 chamber incubation tray insert the tip of forceps into the indented numeral end and gently pry the array upward see figure 2 Using a gloved hand pick up the microarray by holding the array by its edges only Tap to remove excess liquid from slide surface 6 Proceed immediately to Probing the Array Continued on next page 76 Tritium Radiolabeled Tritium Radiolabeled Small Molecule Interaction Probing Procedure Continued Probing the Array 1 10 11 12 Place each microarray horizontally in a separate sterile 50 mL conical tube with about 1 3 of the array extended outside of the tube as shown in the figure below The barcoded end of the array should protrude from the tube face up gs i For each ProtoArray Protein Microarray add 100 pL of probing mixture including the H labeled compound of interest and the positional mapping reagent H estradiol and pipet the mixture gently onto the surface of the ProtoArray Protein Microarray Note Optimal probing concentratio
38. to starting the probing procedure Blocking Step Instructions for blocking the microarray are described below 1 Immediately place the mailer containing the ProtoArray Human Protein Microarray v5 0 at 4 C upon removal from storage at 20 C and equilibrate the mailer at 4 C for at least 15 minutes prior to use 2 Place ProtoArray Human Protein Microarrays with the barcode facing up in the bottom of a 4 chamber incubation tray such 1b x that the barcode end of the microarray is near the tray end containing an indented numeral see figure 1a The indent in the tray bottom is used as the site for buffer removal see figure 1b arrow 3 Using a sterile pipette add 5 mL Blocking Buffer into each chamber Avoid pipetting buffer directly onto the array surface 4 Incubate the tray for 1 hour at 4C on a shaker set at 50 rpm circular shaking 5 After incubation aspirate Blocking Buffer by vacuum or with a pipette Position the tip of the aspirator or pipette into the indented numeral and aspirate the buffer from each well see figure 2 Tilt the tray so that any remaining buffer accumulates at the end of the tray with the indented numeral Aspirate the accumulated buffer Important Do not position the tip or aspirate from the microarray surface as this can cause scratches Immediately proceed to adding the next solution to prevent any part of the array surface from drying which may produce high or uneven background
39. to the protein probe can be used for detection but optimal conditions may need to be independently developed Primary antibodies can be labeled using the Alexa Fluor 647 Protein Labeling Kit Invitrogen Cat no A 20173 Prepare 5 mL of antibody or streptavidin solution for each array to be probed e Primary biotin detection Prepare 1 pg mL Streptavidin Alexa Fluor 647 Conjugate in Washing Buffer e Primary V5 epitope detection Prepare 1 pg mL Alexa Fluor 647 Anti V5 Antibody in Washing Buffer e Secondary V5 epitope detection e Use 1 pg mL Anti V5 Antibody in Washing Buffer for primary antibody e Use 1 pg mL Alexa Fluor 647 Goat Anti Mouse diluted to 1 ug mL in Washing Buffer for secondary antibody ProtoArray Control Protein Microarray The Array Control Protein is detected using a primary or secondary fluorescent conjugate Prepare 5 mL of antibody or streptavidin solution for each array to be probed e Primary detection Prepare 1 ug mL Streptavidin Alexa Fluor 647 Conjugate or Alexa Fluor 647 Anti V5 Antibody in Washing Buffer e Secondary V5 epitope detection e Use 1 pg mL Anti V5 Antibody in Washing Buffer for primary antibody e Use 1 pg mL Alexa Fluor 647 Goat Anti Mouse diluted to 1 pg mL in Washing Buffer for secondary antibody Continued on next page 23 Protein Protein Interaction Probing Procedure Continued Important Before Starting Blocking Step 24 Since most of the human proteins
40. x 10 Example For a kinase 50 000 Da at a protein concentration of 0 5 mg mL the uM protein concentration is uM 0 5 mg mL x 1 50 000 x 10 uM 10 Continued on next page Kinase Substrate Identification Probing Procedure Continued Preparing the Kinase Before Starting You need 120 pL Kinase Buffer with 1 mM DTT containing the Control Kinase or your kinase to probe one ProtoArray Microarray Note Prepare dilutions of the kinase in the Kinase Buffer Component Control Kinase User Kinase Kinase 50 nM 50 nM Kinase Buffer with 1 mM DTT to 120 pL to 120 pL Mix well do not vortex and store on ice until use Immediately return the remaining kinase to 80 C Note Concentration is influenced by activity of kinase and level of kinase autophosphorylation Too much kinase may result in a high background or dark ProtoArray Protein Microarray and too little kinase will result in no additional spots relative to a kinase free control ProtoArray Protein Microarray kinase buffer and y P ATP lacking kinase e Before starting the probing procedure make sure you have all items on hand especially buffers see pages 43 44 kinase in Kinase Buffer see above and coverslips e Make sure the kinase in Kinase Buffer and Kinase Buffer are cold and stored on ice until use Place a 50 mL conical tube on ice to chill the tube until use e Do not store the 0 5 SDS solution on ice Store the 0 5 SDS solution at room
41. 0 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech support amp invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com Certificate of Analysis MSDS Limited Warranty The Certificate of Analysis provides detailed quality control information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box Material Safety Data Sheets MSDSs are available on our website at www invitrogen com msds Invitrogen a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s liability to only the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions The Company reserves the right to select the method s use
42. 0 mM HEPES pH7 5 200 mM NaCl 0 08 Triton X 100 25 Glycerol 20 mM Reduced glutathione 1 mM DTT 1 BSA 1 2 3 4 Prepare 5 mL of buffer for each microarray For 100 mL Blocking Buffer prepare fresh reagents as follows 1M HEPES pH7 5 5mL 5M NaCl 4mL 10 Triton X 100 800 uL 50 Glycerol 50 mL Glutathione powder 610 mg Adjust pH to 7 5 with NaOH Add 100 pL of 1 M DTT and 1 67 mL of 30 BSA prior to use Fill to 100 mL with deionized water Mix well do not vortex and store on ice until use Note Do not store Blocking Buffer containing BSA for more than 24 hrs Assay Buffer final concentration 50 mM Tris HCI pH 7 5 50 mM NaCl 5 mM MgSO 0 1 Tween 20 1 mM DTT 1 BSA 1 Prepare 50 ml of buffer for each microarray For 1 000 mL Assay Buffer prepare fresh reagents as follows 1M Tris HCl pH 7 5 50 mL 5M NaCl 10mL 1 M MgSO 5mL 10 Tween 20 10 mL Add 1 mL of 1 M DTT and 33 3 mL 30 BSA prior to use Fill to 1 000 mL with deionized water Mix well do not vortex and store on ice until use Continued on next page Ubiquitin Ligase Probing Procedure Continued Before Starting Important Blocking Step Before starting the probing procedure make sure you have all items on hand especially buffers see page 88 probes LifterSlip coverslips see page 86 and incubation tray see page 86 Make sure the buffers except are cold Store buffers on ice until use Place an incuba
43. 5 0 KSI Kit 1 kit PAH0525065 for kinase substrate identification 10X Synthetic Block 75 mL PAO17 Blocking Buffer Kit 1 kit PA055 Array Control Protein 40 uL 451096 Alexa Fluor 647 Anti V5 Antibody for ProtoArray 80 uL 451098 Streptavidin Alexa Fluor 647 Conjugate 2 mg mL 0 5 mL S 32357 Control Kinase MAPK14 Active 10 pg PV3304 Biotin XX Microscale Protein Labeling Kit and FluoReporter 1 kit B30756 Biotin Quantitation Assay Kit Alexa Fluor 647 Protein Labeling Kit 1 kit A 20173 Alexa Fluor 647 Goat Anti Mouse IgG H L 0 5 mL A 21236 Alexa Fluor 647 Goat Anti Human IgG H L 0 5 mL A 21445 Anti V5 Antibody 50 pL R960 25 Anti V5 HRP Antibody 50 pL R961 25 Anti V5 AP Antibody 50 pL R962 25 Phosphate Buffered Saline PBS 1X 500 mL 10010 023 ProQuest Two Hybrid System 1 kit PQ10002 01 ProQuest Two Hybrid System with Gateway Technology 1 kit PQ10001 01 Continued on next page 135 Accessory Products Continued Vectors Accessing Clones 136 A variety of vectors with different tags at the N or C terminus is available for expression and purification of your protein of interest The recommended tag for use with the ProtoArray Human Protein Microarray is the V5 epitope tag For more information about these products visit www invitrogen com or call Technical Support page 137 Since the majority of human proteins printed on the array are derived from the Ultimate ORF Clone Collection or purified proteins
44. Array not dried properly Dry the array as described on page 65 before before scanning scanning High probe concentration Decrease the probe concentration or decrease the incubation time Uneven blocking or washing Improper washing During the blocking or washing steps ensure the array is completely immersed in SMI Assay Buffer and use at least 5 mL buffer in the incubation tray to immerse the array completely with buffer To obtain the best results perform the recommended washing steps Prepare the SMI Assay Buffer fresh as described on page 60 Portions of array have dried Do not allow the array to dry during probing Improper array handling Protein probe not applied properly Always wear gloves and avoid touching the surface of the array with gloved hands or forceps Take care while inserting the array into the incubation tray to avoid scratching the array surface Apply the probe solution and LifterSlip or equivalent coverslip to the array as described in the manual To avoid drying of the array surface make sure the coverslip covers the printed area of the array and adjust the coverslip if needed Probe or detection reagents Centrifuge the probe or detection reagents to contain precipitates remove precipitates prior to probing the array Continued on next page Tritium Radiolabeled Small Molecule Identification SMI Radioactive Application Experimental Overview Experimental The experimen
45. E coli V5 Epitope Tag The V5 epitope tag is a 14 amino acid GKPIPNPLLGLDST epitope derived from the P and V proteins of the paramyxovirus SV5 Southern et al 1991 The V5 tag is expressed as a fusion to calmodulin kinase protein and is useful in detection of the protein The Anti V5 Antibody available from Invitrogen page 135 recognizes the 14 amino acid sequence and allows detection of Array Control Protein containing the V5 epitope BioEase tag TM The BioEase tag is a 72 amino acid peptide derived from the C terminus amino acids 524 595 of the Klebsiella pneumoniae oxalacetate decarboxylase a subunit Biotin is covalently attached to a single biotin binding site lysine 561 of the oxalacetate decarboxylase a subunit Schwarz et al 1988 When fused to the Array Control Protein the BioEase tag is sufficient to facilitate in vivo biotinylation of the protein by E coli cellular biotinylation enzymes The Array Control Protein interaction is detected using a streptavidin detection system For control KSI experiments using the ProtoArray Control Protein Microarray a Control Kinase is required The Control Kinase is available from Invitrogen page 135 You can also probe the Control Microarray using your kinase of interest See page 40 for probing options The Control Kinase is a recombinant human MAPK14 p38 alpha purified from insect cells Methods Before Starting Introduction Important Guidelines
46. ENO 7 O E Nous l Antibody Concentration 112 Follow the guidelines listed below for buffer preparation to obtain the best results with microarrays The buffer recipes are listed on the next page e Always use ultra pure water to prepare reagents and buffers e You may use non ionic detergents and reducing agents during probing to minimize non specific interactions e If the protein interaction requires certain co factors be sure to include the co factors in the probing buffer during probing e Prepare the Blocking Buffer and Washing Buffer fresh prior to use e Use the recipes described below to prepare your own buffers Recommended buffers are listed below for blocking and washing the arrays You can perform array probing using the recommended buffers and then based on your initial results optimize the buffer formulation e The recommended primary antibody concentration range for probing each array is 0 1 10 pg mL Dilute concentrated antibody in Washing Buffer e Secondary Alexa Fluor 647 conjugates should be diluted to 1 ug mL in Washing Buffer Continued on next page Antibody Specificity Profiling Application Probing Procedure Continued Preparing Blocking Buffer Preparing Washing Buffer Blocking Buffer final concentration 50mM HEPES pH 7 5 200 mM NaCl 0 08 Triton X 100 25 Glycerol 20 mM Reduced Glutathione 1 mM DTT 1X Synthetic Block 1 Prepare 5 mL of buffer for each m
47. Immune Response Biomarker Profiling Probing Procedure sss 99 Scanning and Data Analysis sse tenente nennen 105 Expected Results for IRBP se er OR e e etae peel a eene aa ea 106 Troubleshooting isss esenee ee edere o ene Up DI UP ey e e ie qas 107 Antibody Specificity Profiling Application eese eese eene teen ennt tentent tn tnn 109 Experimental Overview eene ee neret deer ie e ese ide deese teft hoe 109 Guidelines for Probing the ProtoArray Microarray eese nete rte tette tete tenententnne 110 Antibody Specificity Profiling Application Probing Procedure sess 111 Scanning and Data Analysis siine ieena esek apee R a ana ae KEES a eE EEEE 118 Scanning and Data Analysis Continued sss 119 Expected Results for Antibody Specificity Profiling Applicaton sssssssssssss 120 Trouble shooUng c ect ete e eh e epe d n eie pde teta ite Eo eda 121 Scanning Arrays Using a Fluorescence Scanner eese entente nns 123 Data Acquisition and Analysis tenente tenete tenen 126 Image Acquisition and Processing for Radioactive Assays eese 129 Data Acquisition and AN lySiS sona e tenete tenente 131 PADD OI GIK emm 135 Accessory Products ee e e ehe e eme p e deeft ens 135 Technical Supports s ese b A RR ERI AS HR RU Roe eti aee o meds 137 Purchaser Notificati ni dis Spade pee ug p eH pe RH RER
48. Invitrogen ProtoArray Applications Guide General information technology overview and applications using the ProtoArray Human and Control Protein Microarray Rev date 28 January 2010 Manual part no 25 1025 ii Table of Contents General Information eee E eem eee can use mn pipe eite e preter SET v Introduction E 1 QUEL VIEW ani aE A REE RE E aioe te on eds E estes 1 ProtoArray Human Protein Microarray aset dades absit e a putt et 4 ProtoArray Control Protein Microarray oo eir Pobanptesniqe de pole puli us do FH EIOG 7 MethodS pe 11 Betore Starting c so det ete punto ee tetris rte ip eda tte t E ats 11 Protein Protein Interaction PPI Application eese eee tnne ntn tn tne tn tnn 12 Experimental Ovetview essent eet eei t a iet i tese e HER I Erainn 12 Guidelines for Probing the ProtoArray Microarray eese tette tte testet tente tase 14 Preparing the Protein Probe netten tenente nennen 18 Protein Protein Interaction Probing Procedure sse 20 Scanning and Data Analysis sse nter nennen 28 Scanning and Data Analysis Continued sse 29 Expected Results for PPI smooth e ao Rea eSEE SEKERA E EEEE 30 Troubl shootng susce citet tun Eodem a E emn nts 32 Kinase Substrate Identification KSI Application eese eene tnnt tnn tn antn 35 Experimental Ovetview 5 esed hte He ett en eR AH et iH b re
49. L Kinase Buffer containing 50 nM either your kinase of interest or 50 nM Control Kinase with 33 nM y P ATP Step 2 Using forceps carefully lay a glass coverslip on the surface of the array without trapping any air bubbles Align the coverslip flush with the top edge of the array to ensure the printed area of the array is completely covered If necessary gently adjust the coverslip to remove any air bubbles Gently slide each array with a coverslip into the conical tube with the printed side barcode of the array facing up Cap the conical tube Place each conical tube horizontally on a flat surface in an incubator set to 30 C such that the printed side of the array is facing up and the tube is as level as possible If needed tape the tube to the flat surface to avoid any accidental disturbances Incubate the array in the tube for 1 hour at 30 C without shaking Remove the tubes from the incubator Using a sterile pipette add 40 mL 0 5 SDS page 43 by dispensing the SDS down the sides of the tube Avoid pipetting SDS directly onto the array surface Continued on next page 47 Kinase Substrate Identification Probing Procedure Continued Probing the Array Continued Drying and Exposing the Array 48 Protocol continued from the previous page 9 10 11 12 13 14 15 16 17 18 19 Incubate the array in SDS for 1 minute at room temperature without shaking Gently move the array in the t
50. Materials Needed e ProtoArray Human or Control Protein Microarray page 135 e Buffers see next page e Small molecule probe containing a suitable tag in SMI Assay Buffer next page e Alexa Fluor 647 conjugated streptavidin or equivalent page 135 keep on ice in dark until immediately before use if using biotinylated small molecule e Antibody against the epitope tag for an epitope tagged small molecule probe e Ice bucket e Deionized water e Clean covered 4 chamber incubation tray Greiner Cat no 96077307 chilled on ice e LifterSlip coverslips Thermo Scientific Cat no 25x601 2 4789 e Microarray slide holder and centrifuge equipped with a plate holder Optional Incubation Trays The microarray is placed in an incubation tray during the blocking and washing steps To obtain the best results all incubations of the ProtoArray with various solutions are performed in a 4 chamber covered incubation tray Greiner Cat no 96077307 Coverslips LifterSlip coverslips Thermo Scientific Cat no 25x601 2 4789 hold a small reagent volume to minimize the amount of valuable probe used and prevent evaporation of reagents If you are using any other coverslip be sure the coverslip is able to completely cover the printed area 20 mm x 60 mm of the glass slide and the coverslip is made of non protein binding material Untreated glass coverslips are not recommended Continued on next page 59 Small Molecule Interact
51. Microarray using buffer recipes provided in this manual see page 74 for buffer recipes 1 Block the ProtoArray Human Protein Microarray 2 Probe with your radiolabeled small molecule probe 3 Perform detection using an appropriate detection system 5 Dry the array for exposing ProtoArray Human or Control Protein Microarray page 135 Tritium SMI Assay Buffer page 74 Estradiol 2 4 6 7 16 17 H N Perkin Elmer Cat no NET517 Radiolabeled small molecule probe containing a suitable tag in Tritium SMI Assay Buffer see next page Ice bucket Deionized water Clean covered 4 chamber incubation tray Greiner Cat no 96077307 chilled on ice Coverslips VWR Cat no 48404 454 Exeter Conservation Board Light Impressions 3500 or thick filter paper Microarray slide holder and centrifuge equipped with a plate holder Optional You will need coverslips that are able to completely cover the printed area 20 mm x 60 mm of the glass slide and hold a small reagent volume to minimize the amount of valuable probe used and prevent evaporation of reagents We recommend using glass coverslips VWR Cat no 48404 454 Continued on next page 73 Tritium Radiolabeled Small Molecule Interaction Probing Procedure Continued Using Your Own Buffers Nds by NY A B v fes o 2 gt Preparing Tritium SMI Assay Buffer 74 Follow the guidelines listed below for buffer preparation to obtain the best results with
52. O EARE irt rir iei e ids 138 References ih saci Ustad tail mai dai nant ierit ato id var Hu ee e 140 General Information Purpose of the Guide Shipping and Storage Contents Intended Use The ProtoArray Applications Guide contains information about the ProtoArray Human and Control Protein Microarrays The ProtoArray Applications Guide includes the following information e ProtoArray technology overview e Description of the ProtoArray Microarray e General guidelines for using the ProtoArray Microarray e Protocol to perform Protein Protein Interactions PPI application e Protocol to perform Kinase Substrate Identification KSI application e Protocol to perform Small Molecule Protein Interaction SMI profiling application for biotinylated fluorescently labeled and radiolabeled small molecules e Protocol to perform Immune Response Biomarker Profiling IRBP application e Protocol to perform Ubiquitin ligase profiling application e Protocol to perform Antibody Specificity Profiling ASP application e Scanning and data analysis e Examples of expected results e Troubleshooting Each ProtoArray Human or Control Microarray is shipped on blue ice Upon receipt store the microarray at 20 C An expiration date is printed on the packaging for the microarray Use the array before expiration for best results Each ProtoArray Microarray Box contains a mailer with one ProtoArray Human or Control Protein Microar
53. PLA 71 Guidelines for Probing the ProtoArray Microarray esee ttntte tinte tete ntnn 72 Tritium Radiolabeled Small Molecule Interaction Probing Procedure sss 73 Image Acquisition and Processing sse tenete nennen 79 lii Expected Results for SMI Radioactive c ccccccccsccsesesssssnsessseseeceeesenenesesesesssnsnenesessseseeceeeensseseeeeeenanenes 81 Troubleshooting sss so ooi iet Maree o oit so Md sin Get dpi ted ose oul sitesi Ge 82 Ubiquitin Ligase Profiling Application eese ertet enne ne tn tntnstn etna tns tn etna tn snin 84 Experimental OVerview 2e eher ttes iae aeeaiei aeae heredis ettari so cenfeo iege 84 Guidelines for Probing the ProtoArray Microarray esee tstete tn tente tatnen ta stent se 85 Ubiquitin Ligase Probing Procedure sse tenete 86 Scatining and Data Analysis 53 e eR D P en d ted et a Een 92 Scanning and Data Analysis Continued sse tenerent 93 Expected Results for Ubiquitin Ligase ise poitin anane kie a KEE no eenia ak nnne 94 Troubleshooting eo setis eest Enon ve pe eet te e HL Pe ie aa eme HEU SERERE PEL ARE 95 Immune Response Biomarker Profiling IRBP Application eere 97 Experimental Overview usSesebiedebeeuio scd eiie tec e red e ERR RE HERR ERR 97 Guidelines for Probing the ProtoArray Microarray esee tenete tete tte tatnen 98
54. Processing The ProtoArray Prospector Imager allows image processing for data analysis Using ProtoArray Prospector Imager Image Processing Using Adobe Photoshop 130 1 N Go to www invitrogen com protoarray and then click on the Online Tools link to download and install the ProtoArray Prospector installation package including ProtoArray Prospector Imager Start ProtoArray Prospector Imager on the computer Open the microarray image tiff acquired in Step 4 previous page Perform the following adjustments to the image refer to ProtoArray Prospector Imager manual for detailed instructions e Invert the data convert the image from white background with black spots to black background with white spots which is required for analysis e Rotate the image such that the array image is vertical and the barcode is located at the bottom e Crop a fixed rectangular area 600 x 1800 pixel if scanned at 600 dpi from each image tiff file corresponding to the array If the spots are not aligned vertically rotate the crop rectangle by holding the Ctrl key and rotating the selection angle with the mouse First rotate and align the rectangle against the Alignment Control Kinase PKCeta spots release the Ctrl key and move the rectangle to cover the whole array area Crop the image using the Crop button If needed adjust the image contrast brightness in Imager for better visualization which will not affect the f
55. Production of Biantennary Complex N glycans J Biochemistry 41 15093 15104 Jin F T H A M G M S F P P S F and J H 2006 A Pooling deconvolution Strategy for Biological Network Elucidation Nature Methods 3 161 162 Mah A S Elia A E Devgan G Ptacek J Schutkowski M Snyder M Yaffe M B and Deshaies R J 2005 Substrate Specificity Analysis of Protein Kinase Complex Dbf2 Mob1 by Peptide Library and Proteome Array Screening BMC Biochem 6 22 33 Mattoon D Michaud G Merkel J and Schweitzer B 2005 Biomarker Discovery Using Protein Microarray Technology Platforms Antibody antigen Complex Profiling Expert Rev Proteomics 2 879 889 Michaud G A Salcius M Zhou F Bangham R Bonin J Guo H Snyder M Predki P and Schweitzer B 2003 Analyzing Antibody Specificity With Whole Proteome Microarrays Nature Biotechnol 21 1509 1512 Predki P 2003 Functional Protein Microarrays Ripe for Discovery Curr Opin Chem Biol 8 8 13 Ptacek J Devgan G Michaud G Zhu H Zhu X Fasolo J Guo H Jona G Breitkreutz A Sopko R McCartney R Schmidt M Rachidi N Lee S J Mah A Meng L Stark M Stern D De Virgilio C Tyers M Andrews B Gerstein M Schweitzer B Predki P and Snyder M 2005 Global Analysis of Protein Phosphorylation in Yeast Nature 438 679 684 Satoh J Nanri Y and Yamamura T 2006 Rapid Identific
56. SMI Assay Buffer Mix well do not vortex and store on ice until use 5H Estradiol Add the positional mapping reagent H Estradiol to 100 uL of your small molecule probe at a final concentration of 40 pCi pL e Before starting the probing procedure make sure you have all items on hand especially buffers previous page probes in Tritium SMI Assay Buffer previous page and coverslips e Make sure the buffers are cold Store buffers on ice until use e Review Important Guidelines on page 11 and Working with Radioactive Material on page 38 prior to starting the probing procedure Incubation chambers are not suitable for use in the probing portion of the SMI radioactive application A container that seals tightly is required to prevent any leakage of radioactive material during the washing steps Continued on next page 75 Tritium Radiolabeled Tritium Radiolabeled Small Molecule Interaction Probing Procedure Continued Blocking Step Instructions for blocking the microarray are described below 1 Immediately place the mailer containing the ProtoArray Human Protein Microarray v5 0 at 4 C upon removal from storage at 20 C and equilibrate the mailer at 4 C for at least 15 minutes prior to use be sure to use the microarray before the expiration date printed on the box 2 Place ProtoArray Human Protein Microarrays with the barcode facing up in the bottom of a 4 chamber incubation tray such that the barcode end of
57. We recommended GenePix Pro v6 or later Molecular Devices Corporation or ScanArray Acquisition Software PerkinElmer Inc as microarray data acquisition software for analysis of images For detailed instructions on scanning the microarray refer to Scanning Arrays Using a Fluorescence Scanner page 123 7 Insert array into the fluorescence microarray scanner 8 Adjust scanner settings 9 Preview the microarray and adjust settings if needed 10 Scan the microarray 11 Save image data 12 Export and analyze results For detailed instructions on Data Acquisition and Analysis refer to page 126 4 To acquire data from the scanned image use the barcode on the array to download the GAL file from ProtoArray Central as described on page 126 5 Use the GAL file and suitable microarray data acquisition software to acquire pixel intensity values for all features on the array 6 Analyze data with ProtoArray Prospector using the guidelines on page 128 to determine significant signals with the controls and your protein probe Continued on next page Scanning and Data Analysis Continued Analyzing ProtoArray Prospector Results The Next Step After data analysis ProtoArray Prospector presents a summary of the analyzed data in a table format see ProtoArray Prospector manual for details The proteins that score as positive in the experiment are proteins that satisfy the basic program options Review the inform
58. al dilutions are performed correctly Protein has low lysine content or lysine residues are not readily available for biotinylation Perform the biotinylation reaction at a higher molar ratio You may express your protein as fusion to a tag that contains lysine Protein impurities present that undergo biotinylation and may cause high background during probing Purify protein to remove impurities and perform biotinylation to ensure the absence of additional biotinylated bands Continued on next page Troubleshooting Continued Protein Array Results Weak or no signal Epitope tag not present or Confirm the presence of the tag by western with protein probe not accessible detection Ensure the tag is accessible under native conditions by performing an ELISA Poor biotinylation of protein See previous page for details probe Low probe concentration Perform probing with higher probe concentration or increase the incubation time Incorrect probing procedure Follow the recommended protocol for probing on page 26 Be sure all incubations are performed at 4 C Prepare the Blocking Buffer and Washing Buffer fresh as described on page 21 22 Do not allow the array to dry during the probing procedure Avoid prolonged exposure of detection reagents labeled with a fluorescent dye to light Incorrect scanning or Scan the array at suitable wavelength for the imaging detection system used and place the array in the slide holde
59. and analyze data Protein probe or detection system reacting with array chemistry High Low Decrease probe Yes background background concentration Increase stringency Yes Increase probe gt 1 Protein probe and concentration Expected protein No inter ction with detection system interaction observed negative control s Protein not functional on with protein probe or observed arrays positive control compatible for use with protein microarray 17 Preparing the Protein Probe Introduction Protein Tags Generating Tagged Protein Probe 18 Before using the ProtoArray Human Protein Microarray you need your purified protein of interest to probe the microarray The protein of interest must contain a suitable tag see below You may purify proteins using a method of choice You can use proteins purified from E coli yeast cells or higher eukaryotes to probe the ProtoArray Human Protein Microarray The amount of protein and quality of protein required for probing are described in this section The protein of interest can be tagged using an epitope tag or a biotin label Using an epitope tag at the N or C terminus of the probe allows the use of the recombinant fusion protein directly as a probe without any further modification wherein the tag is used as the marker for detection of interactions The recommended epitope tag is the V5 epitope tag at the N or C terminus of
60. ately in blocking solution equilibrated at 4 C Condensation on the array may reduce protein activity or alter spot morphology e Perform array experiments at a clean location to avoid dust or contamination Filter solutions if needed Particles invisible to the eye can produce high background signals and cause irregular spot morphology e Donot allow the array to dry out during the experiment Cover the array completely with the appropriate reagent during all steps of the protocol e Always dry the array prior to scanning Scan the array on the same day at the end of the experiment e Do not dry the array using compressed air or commercial aerosol sprays 11 Protein Protein Interaction PPI Application Experimental Overview Experimental The experimental outline for performing the PPI application using the Outline ProtoArray Human Protein Microarray v5 0 to identify potential protein protein interactions is described below See next page for the experimental workflow Step Action Page no 1 Express your protein of interest as a fusion protein in an expression vector containing the desired tag at the N or C terminus of the protein and purify the protein OR In vitro biotinylate your protein of interest using a method of choice 18 Block ProtoArray Human Protein Microarray with 5 mL Blocking Buffer 24 Probe the ProtoArray Human Microarray with the protein probe and perform detection using a s
61. ation of 14 3 3 binding Proteins by Protein Microarray Analysis J Neurosci Methods 152 278 288 Schwarz E Oesterhelt D Reinke H Beyreuther K and Dimroth P 1988 The Sodium Ion Translocating Oxalacetate Decarboxylase of Klebsiella pneumoniae J Biol Chem 263 9640 9645 Schweitzer B Predki P and Snyder M 2003 Microarrays to Characterize Protein Interactions on a Whole Proteome Scale Proteomics 3 2190 2199 Southern J A Young D F Heaney F Baumgartner W and Randall R E 1991 Identification of an Epitope on the P and V Proteins of Simian Virus 5 That Distinguishes Between Two Isolates with Different Biological Characteristics J Gen Virol 72 1551 1557 Zhu H Bilgin M Bangham R Hall D Casamayor A Bertone P Lan N Jansen R Bidlingmaier S Houfek T Mitchell T Miller P Dean R A Gerstein M and Snyder M 2001 Global Analysis of Protein Activities Using Proteome Chips Science 293 2101 2105 2010 Life Technologies Corporation All rights reserved Trademarks referenced herein are registered trademarks or trademarks of Life Technologies Corporation Any registration or trademark symbols used herein denote the registration status of trademarks in the United States Trademarks may or may not be registered in other countries Adobe Photoshop is a registered trademark Adobe Systems Incorporated Alphalmager is a trademark of Alpha Innotech Corporation Coo
62. ation on pages 68 Expected Results to help with data interpretation We recommend validating the interactions as described below After identifying a positive interaction on the ProtoArray Human Protein Microarray you may validate the small molecule interaction using the ProtoArray Technology or other methods Using the ProtoArray Technology validate the small molecule interactions by performing experiments with additional arrays to ensure e Reproducibility Probe protein arrays using a similar or a different probe concentration to observe similar interactions e Specificity Probe protein arrays with the detection reagent used to visualize the interactions and also different small molecules containing the tag to identify interactions specific to your small molecule probe of interest and also identify any non specific interactions In addition competition assays may be performed to determine if the interactions can be competed by excess unlabeled small molecule OR e Interactions observed on the ProtoArray Human Protein Microarray can be validated using solution phase assays Continued on next page 67 Expected Results for SMI Fluorescent Control Array Results obtained after probing the ProtoArray Control Protein Microarray v5 0 Probing Results with Alexa Fluor 647 labeled staurosporin a known binding partner for calmodulin kinase is shown below Control Array probed with Alexa Fluor 647 labeled Sta
63. ay Buffer with gentle agitation for 5 minutes Aspirate SMI Assay Buffer Repeat wash step three times Add 5 mL streptavidin Alexa Fluor 647 diluted in SMI Assay Buffer Add streptavidin Alexa Fluor 647 at the indented numeral end of the 4 well tray and allow the liquid to flow across the slide surface To prevent local variations in fluorescence intensity and background avoid direct contact with the slide Incubate for 30 minutes at 4 C with gentle shaking 50 rpm Remove streptavidin Alexa Fluor 647 solution by aspiration Wash with 5 mL SMI Assay Buffer with gentle agitation for 5 minutes Aspirate SMI Assay Buffer Repeat wash step three times Remove the array from the 4 well tray using forceps Proceed to Drying the Array Continued on next page Small Molecule Interaction Probing Procedure Continued Drying the Array 1 To remove the array from the 4 chamber incubation tray insert the tip of forceps into the indented numeral end and gently pry the array upward see Step 1 page 64 Using a gloved hand pick up the microarray by holding the array by its edges Place the array in a slide holder or a sterile 50 mL conical tube if you do not have a slide holder Ensure the array is properly placed and is secure in the holder to prevent any damage to the array during centrifugation Briefly dip the slide holder containing the arrays into room temperature distilled water three times to remove salts If you are not
64. ay performance with the optional blocking reagents is small molecule specific and can be determined through pilot experiments on Control arrays To prepare 1 Casein dissolve the casein in Tritium SMI Assay Buffer and heat solution at 50 C until casein is completely dissolved Do not exceed 60 C Do not microwave Continued on next page Tritium Radiolabeled Small Molecule Interaction Probing Procedure Continued Preparing Small Molecule Probes Before Starting Important ProtoArray Human Protein Microarray To probe the microarray you need 100 uL of your tritiated small molecule probe for each array The recommended activity range for the final concentration of your small molecule probe is 50 pCi L 50 nCi pL with weaker interactions requiring an activity of 10 50 nCi pL We recommend that the tritiated small molecule stock activity be at least luCi uL with a specific activity of at least 10 Ci mmol and that a minimum of 60 pCi be available to perform each small molecule protein interaction experiment If the tritiated small molecule is dissolved in an organic solvent such as ethanol or DMSO the final organic solvent concentration should be less than 1 DMSO by volume or 5 ethanol by volume To avoid non specific interactions and or high background we further recommend that the final concentration of tritiated small molecule be no higher than 1 uM Dilute the probe to the recommended starting concentration in Tritium
65. bucket e Forceps and deionized water e 10X Synthetic Block see page 135 e Clean covered 4 chamber incubation tray Greiner Cat No 96077307 chilled on ice e LifterSlip coverslips Thermo Scientific Cat No 25X601 2 4789 e Shaker capable of circular shaking at 50 rpm place the shaker at 4 C e Microarray slide holder and centrifuge equipped with a plate holder Optional Incubation Trays The microarray is placed in an incubation tray during the blocking and washing steps To obtain the best results all incubations of the ProtoArray with various solutions are performed in a 4 chamber covered incubation tray Greiner Cat no 96077307 Coverslips LifterSlip coverslips Thermo Scientific Cat no 25X601 2 4789 hold a small reagent volume to minimize the amount of valuable probe used and prevent evaporation of reagents If you are using any other coverslip be sure the coverslip is able to completely cover the printed area 20 mm x 60 mm of the glass slide and the coverslip is made of non protein binding material Untreated glass coverslips are not recommended Continued on next page 20 Protein Protein Interaction Probing Procedure Continued Using Your Own Buffers Preparing Blocking Buffer If you are preparing your own buffers follow the guidelines listed below for buffer preparation to obtain the best results with microarrays The buffer recipes are listed below e Always use ultra pure water to
66. d equilibrate the mailer at 4 C for at least 15 minutes prior to use 2 Place ProtoArray Human Protein Microarrays with the barcode facing up in the bottom of a 4 chamber incubation tray such that the barcode end of the microarray is near the tray end containing an indented numeral see figure 1a The indent in the tray bottom is used as the site for buffer removal see figure 1b arrow 3 Using a sterile pipette add 5 mL Blocking Buffer into each chamber Avoid pipetting buffer directly onto the array surface 4 Incubate the tray for 1 hour at 4 C on a shaker set at 50 rpm circular shaking Use a shaker that keeps the arrays in one plane during rotation Rocking shakers are not to be used because of increased risk of cross well contamination 5 After incubation aspirate Blocking Buffer by vacuum or with a pipette Position the tip of the aspirator or pipette into the indented numeral and aspirate the buffer from each well see figure 2 Tilt the tray so that any remaining buffer accumulates at the end of the tray with the indented numeral Aspirate the accumulated buffer Important Do not position the tip or aspirate from the microarray surface as this can cause scratches Immediately proceed to adding the next solution to prevent any part of the array surface from drying which may produce high or uneven background 6 Proceed immediately to Probing the Array 115 Antibody Specificity Profiling Application Pr
67. d of the microarray is near the tray end containing an indented numeral see figure 1a The indent in the tray bottom E is used as the site for buffer removal see figure 1b arrow Using a sterile pipette add 5 mL Blocking Buffer into each chamber Avoid pipetting buffer directly onto the array surface Continued on next page 89 Ubiquitin Ligase Probing Procedure Continued Blocking Step Instructions for blocking the microarray are described below continued 4 Incubate the tray for 1 hour at 4 C on a shaker set at 50 rpm circular shaking Use a shaker that keeps the arrays in one plane during rotation Rocking shakers are not to be used because of increased risk of cross well contamination 5 Prepare 100 uL of ubiquitin ligase probe mixture page 87 and incubate at 30 C for 5 minutes 6 After incubation aspirate Blocking Buffer by vacuum or with a pipette Position the tip of the aspirator or pipette into the indented numeral and aspirate the buffer from each well see figure 2 Tilt the tray so that any remaining buffer accumulates at the end of the tray with the indented numeral Aspirate the accumulated buffer Important Do not position the tip or aspirate from the microarray surface as this can cause scratches Immediately proceed to adding the next solution to prevent any part of the array surface from drying which may produce high or uneven background 7 Proceed immediately to Probing the Array
68. d on the fluorophore used for detection for Alexa Fluor 647 use 635 nm PMT Gain 600 Laser Power 100 Pixel Size 10 uM Lines to Average 1 0 Focus Position 0 uM 6 Performa preview to quickly scan the microarray Adjust the PMT Gain if needed Note The image should have very few saturated white spots to keep the majority of feature signals within the linear range of the scanner 7 Select the area of the array to scan in detail include the barcode in the area for documentation purposes and then scan the array to create a high resolution image 8 After acquiring the image save the image to a suitable location as multi image TIFF file Be sure the barcode is included in the name of the image 9 Open the microarray enclosure and remove the microarray from the holder 10 Proceed to Data Acquisition and Analysis next page 125 Data Acquisition and Analysis Introduction Important GAL File ProtoArray Central 126 After scanning and saving an image of the array download the protein array lot specific information from the ProtoArray Central Portal Use the lot specific information to acquire and analyze the data to identify protein protein interactions Note To familiarize yourself with the array and subarray layout you may download a file showing the subarray layout from ProtoArray Central To access ProtoArray Lot Specific Information see below While downloading the lot specific
69. d to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 137 Purchaser Notification Limited Use Label License No 5 Invitrogen Technology Limited Use Label License No 125 GST 138 The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its componen
70. dditional arrays to ensure e Reproducibility Probe the human array using a similar or a different small molecule concentration to address reproducibility e Specificity Probe protein arrays with different radiolabeled small molecules to identify interactions specific to your small molecule probe of interest and also identify any non specific interactions In addition competition assays may be performed to determine if the interactions can be competed by excess unlabeled small molecule OR e Interactions observed on the ProtoArray Human Protein Microarray can be validated using solution phase assays 80 Expected Results for SMI Radioactive Control Array Probing Results Note Results obtained after probing the ProtoArray Control Protein Microarray v5 0 with H estradiol which binds to Estrogen Receptor alpha is shown below Image showing the Control Array when probed with H estradiol Control Array Boxed area shown in detail Image ER alpha ER alpha we The following control features can be observed after probing a ProtoArray Protein Microarray e Estrogen Receptor ER alpha Estrogen Receptor alpha is spotted twice on each subarray The specific interaction between 5H estradiol and ER alpha indicate that the probing procedure and scanning is performed properly and are used for reference spots to orient the microarray image and help assign spot identities To
71. dulin kinase as the probe to detect the interacting protein calmodulin The ability to observe reciprocal interactions suggests that the proteins maintain a properly folded state on the array Continued on next page 29 Expected Results for PPI Results obtained after probing the ProtoArray Control Protein Microarray v5 0 with the Array Control Protein i e BioEase V5 tagged biotinylated calmodulin Control Array Probing Results kinase are shown below Protein and Streptavi Control Array probed with 50 pg mL Array Control din Alexa Fluor 647 Conjugate Control Array probed with 5 pg mL Array Control Protein and Anti V5 Alexa Fluor 647 Antibody Array image Boxed Area shown in detail Array image Boxed Area shown in detail ES e e Biotin Ab Alexa Fluor Ab Alexa Fluor Ab Alexa Fluor SEPT Calmodulin V5 Control Protein a m Feature is Cmd1p or CALM2 depending on the array lot Refer to the lot specific GAL file for the specific identity of the protein FI Alexa Fluor Ab Alexa Fluor Calmodulin V5 Control Protein DOYYYEXEEN Feature is Cmd1p or CALM2 depending on the array lot Refer to the lot specific GAL file for the specific identity of the protein The following control features can be observed after probing a ProtoArray Protein Microarray e Alexa Fluor Ab signal This is an antibody labeled with Alexa Fluor
72. e ATP mixture added immediately to the immediately add the mixture to the array Do array not store the prepared kinase ATP mixture on ice for more than 2 minutes prior to use on the array Kinase specific substrates Use another kinase are not present on the array Continued on next page 54 Troubleshooting Continued High background Improper blocking Prepare the KSI Blocking Buffer fresh as described on page 43 Improper washing For the best results perform the recommended washing steps using 0 5 SDS and water as outlined in the protocol Array dried during Do not allow the array to dry during probing or probing or washing washing procedure Ensure the coverslip completely covers the printed area of the array During the incubation step at 30 C make sure the 50 mL conical tube is capped to minimize drying During all wash steps ensure the array is completely covered in buffers Array not dried properly Dry the array as described before scanning before scanning High kinase concentration Decrease the kinase concentration specific activity or decrease the incubation time Uneven background Uneven blocking or During the blocking or washing steps ensure the washing array is completely immersed in buffers and use at least 40 mL buffer in the 50 mL conical tube to cover the array completely with buffer Improper washing To obtain the best results perform the recommended washing steps Prepare the 0 5 SDS s
73. e data obtained from different microarrays The ProtoArray Prospector software and manual are available for FREE to ProtoArray users To download the ProtoArray Prospector software and manual go to www invitrogen com protoarray and click on the Online Tools link Install ProtoArray Prospector to install ProtoArray Prospector Imager and Analyzer The ProtoArray Prospector software also accepts the output files GPR generated by the GenePix Pro microarray data acquisition software and analyzes the data using specified algorithms to generate a list of human proteins that bind the small molecule If GPR files are not available consult the ProtoArray Prospector manual for guidelines to format a results file that is compatible for import into Prospector Appendix Accessory Products Additional The table below lists additional products available separately from Invitrogen Products For more information about these products visit www invitrogen com or contact Technical Support page 137 Product Quantity Catalog no ProtoArray Products ProtoArray Human Protein Microarray v5 0 1 array PAH052501 20 arrays PAH0525020 ProtoArray Control Protein Microarray v5 0 1 array PA10057 ProtoArray Human Protein Microarray v5 0 PPI Kit 1 kit PAH0525013 for V5 tagged proteins ProtoArray Human Protein Microarray v5 0 PPI Kit 1 kit PAH0525011 for biotinylated proteins ProtoArray Human Protein Microarray v
74. e initial results with a second array using the same experimental conditions If the initial results indicate weak signal or an unacceptable signal to noise ratio probe a second array with a different probe concentration as described below Probe first array And Then Probe Second Array With 10 nM probe Weak signal With 50 100 nM probe With 50 nM probe High background With 1 10 nM probe Continued on next page 39 Preparing the Protein Kinase Introduction Protein Amount and Quality 40 Before using the ProtoArray Human Protein Microarray for KSI you need to purchase or purify the protein kinase of interest to probe the microarray You may purify the protein kinase using any method of choice You can use proteins purified from E coli yeast cells or higher eukaryotes to probe the ProtoArray Microarray A large variety of highly purified protein kinases are available from Invitrogen For details visit www invitrogen com or contact Technical Support page 137 The amount of protein and quality of protein required for probing are described below e Purify the protein kinase under native conditions e Proteins should be gt 90 pure as determined by Coomassie staining e Check the activity of the protein kinase after purification using a method of choice e Dilute the kinase for use during probing in the Kinase Buffer see recipe on page 44 e Make sure the protein kinase is sol
75. earch button Input Barcode Number s Search 4 For each input barcode the following files are available for downloading GAL file LotNumber gal This file is essential for data acquisition by the software and defines spot locations and identities of all protein spots on the array The file also includes the detected protein concentration information for each spot Protein Information File LotNumber_info txt This file contains a listing and description of the human proteins on the microarray Protein Sequence File LotNumber_seq txt This tab delimited text file lists the GenBank accession number Ultimate ORF Clone ID number if available FASTA header and amino acid sequence of the ORF for each array protein Control Data File LotNumber control txt This file contains a description of control spots on the array Slide Information File LotNumber slide txt This file contains a listing of all barcodes associated with a specific lot of arrays Note The file size for some files such as the Protein Sequence File may be larger than 1 MB Continued on next page Data Acquisition and Analysis Continued Data Acquisition Data acquisition software is used to obtain pixel intensity information for each spot feature on the array Information on additional parameters may be recorded depending on the type of software used for data acquisition 1 Start the ProtoArray Prospector Imager GenePix Pro So
76. eated microarray with denaturing SDS as described in the assay protocol Continued on next page 51 Expected Results for KSI Continued Control Array Probing Results Results obtained after probing the ProtoArray Control Protein Microarray v5 0 with the Control Kinase and radiolabeled ATP are shown below Signal spots without captions represent features exhibiting autophosphorylation Image showing the Control Array when probed with labeled ATP only negative control Image showing the Control Array when probed with 120 nM Control Kinase Control Array Image Boxed area shown in detail Control Array Image Boxed area shown in detail 99 Alignment Control Kinase PKCeta Alignment Control Kinase PKCeta Alignment Control Kinase PKCeta Control Kinase Substrate MAPKAP Alignment Control Kinase PKCeta The following control features can be observed after probing a ProtoArray Protein Microarray e Alignment Control Kinase Signal Alignment Control Kinase PKCeta on the arrays autophosphorylate during the labeling reaction The signals at Alignment Control Kinase locations indicate that the probing procedure and scanning is performed properly and are used as reference spots to orient the microarray image and help assign spot identities e Control Kinase Substrate Signal The Control Kinase substrate is printed on the mic
77. ection e When performing fluorescence detection it is important to avoid exposing the array to light after probing with a fluorescent detection reagent e If performing direct labeling always verify that labeling does not affect the binding affinity of the antibody e Although Alexa Fluor 555 or Cy3 dyes can be used for detection using them may result in higher background signals Continued on next page Guidelines for Probing the ProtoArray Microarray Continued Control Protein Microarray Probing Options Human Protein Microarray Probing Options If you are a first time user of the ProtoArray Human Protein Microarray we recommend that you probe a ProtoArray Control Protein Microarray available from Invitrogen page 135 prior to probing the human microarray The ProtoArray Control Protein Microarray contains various controls and protein interactors printed on the array to allow you to validate probing and detection protocols Probing options can be performed individually or in tandem and include e Probing with the Array Control Protein supplied with the ProtoArray Control Protein Microarray positive control The result from the positive control helps to determine signals specific to your probe e Probing with your protein probe of interest to help you determine background signal and possible array surface interactions e Probing with your biotinylated protein probe to verify level of biotinylation and prese
78. ection reagents contain precipitates reagents to remove precipitates prior to probing the array 108 Antibody Specificity Profiling Application Experimental Overview Experimental The experimental outline for performing antibody specificity profiling service Steps application using the ProtoArray Human Protein Microarray with untagged antibodies and detecting interactions with Alexa Fluor 647 labeled secondary antibody is shown below Step Action Page no 1 Block ProtoArray Protein Microarray with 5 mL Blocking Buffer 115 Probe with 120 uL primary antibody diluted in Washing Buffer with no agitation Optional If you are a first time user of the ProtoArray Human Protein Microarray perform a control probing using a ProtoArray Control Microarray to verify probing and detection protocols 116 Dry the microarray 117 Scan slide with fluorescence microarray scanner 118 Download the protein array lot specific information GAL file from ProtoArray Central Portal to acquire and analyze the data using ProtoArray Prospector to validate antibody specificity 118 Continued on next page 109 Guidelines for Probing the ProtoArray Microarray Human Protein A number of options are available for probing the ProtoArray Human Protein Microarray Microarray with your own buffers and detection reagents as described below Probing Options Review the informati
79. ed anti V5 antibody diluted in Washing Buffer to 0 1 ug mL Signals from the V5 Control Protein gradient printed in each subarray can be used for sample independent external normalization of the IRBP data using the ProtoArray Prospector software see ProtoArray Prospector manual for details Add 5 mL Alexa Fluor 647 antibody solution from Step 8 to the incubation tray at the indented numeral end of the tray without touching the array surface Allow the solution to flow across the array surface Avoid pipetting solution directly onto the array surface Incubate the tray for 90 minutes at 4 C on a shaker set at 50 rpm circular shaking Aspirate the antibody solution as described on the previous page Step 5 Wash each array with 5 mL Washing Buffer with gentle shaking on a shaker set at 50 rpm for 5 minutes at room temperature Aspirate the Washing Buffer as described on the previous page Step 5 Repeat Step 12 four more times using fresh Washing Buffer each time to obtain a total of 5 wash steps Proceed immediately to Drying the Array Continued on next page 103 Immune Response Biomarker Profiling Probing Procedure Continued Drying the Array 104 1 To remove the array from the 4 chamber incubation tray insert the tip of forceps into the indented numeral end and gently pry the array upward see figure below Using a gloved hand pick up the microarray by holding the array by its edges Place the array i
80. elines to format a results file that is compatible for import into ProtoArray Prospector Image Acquisition and Processing for Radioactive Assays Introduction Materials Needed Experimental Outline Scanning Guidelines Once you have exposed the ProtoArray Microarray to X ray film or phosphor screen scan the film or phosphor screen to acquire a TIFF image that is required for microarray data analysis To make the image compatible with the microarray data acquisition software process the image using ProtoArray Prospector Imager or Adobe Photoshop image analysis software as described on the next page Scanning the X ray film You need a standard desktop flatbed image scanner that provides at least 50 uM resolution gt 600 dpi to scan the X ray film after developing the film to produce a 16 bit TIFF files Scanning the Phosphor Screen You need a phosphorimager that provides at least 50 uM resolution to acquire the microarray image from the phosphor screen to produce a 16 bit TIFF file The following phosphorimagers have been tested with the ProtoArray Microarrays e Cyclone Storage Phosphor System PerkinElmer Inc e Typhoon Imager Amersham Biosciences Data acquisition software To acquire ProtoArray data from the image you need ProtoArray Prospector Imager 5 0 or higher The latest version of Prospector Imager is included with ProtoArray Prospector and can be downloaded at www invitrogen com prot
81. equire higher specific activities The radioactivity of the H ligand in the probing solution should be at least 10 50 nCi pL final activity and the H ligand concentration should be 100 nM 1yM in the solution used to probe the arrays Various options are available for performing the probing procedure upon the amount of validation required for probing and detection results If you are a first time user of the ProtoArray Human Protein Microarray we recommend that you probe a ProtoArray Control Protein Microarray available from Invitrogen page 135 prior to probing the human microarray The ProtoArray Control Protein Microarray contains various controls and protein interactors printed on the array to allow you to validate probing and detection protocols Probing options can be performed individually or in tandem and include e Probing with H estradiol positional mapping reagent The result from the positional mapping reagent can serve as a positive control to help determine signals specific to your probe e Probing with your tritiated small molecule of interest to help you determine background signal and possible array surface interactions For details on running a ProtoArray Control Protein Microarray refer to the protocol for the KSI application page 35 The recommended small molecule probe activity range for probing the ProtoArray Human Protein Microarray is 50 pCi pL 50 nCi pL with weaker interactions requiring activity
82. er at 4 C e Microarray slide holder and centrifuge equipped with a plate holder Optional Continued on next page 99 Immune Response Biomarker Profiling Probing Procedure Continued Sample Preparation Incubation Trays Using Your Own Buffers 100 The IRBP application has been optimized for use with human serum and plasma samples fresh or frozen Avoid repeated freeze thaw cycles with samples Prior to use process the sample to remove any aggregates by centrifugation 12 000 x g for 30 seconds on a microcentrifuge if necessary We recommend using a 1 500 dilution of the serum or plasma sample in Washing Buffer to maximize signals while minimizing false positive and false negative results Based on your initial results you may need to optimize the sample dilution to obtain optimal performance To obtain the best results all incubations of the ProtoArray with various solutions are performed in a 4 chamber covered incubation tray Greiner Cat no 96077307 Do not use LifterSlip or any other coverslip for the IRBP application Follow the guidelines listed below for buffer preparation to obtain the best results with microarrays The buffer recipes are listed on the next page e Always use ultra pure water to prepare reagents and buffers e You may use non ionic detergents and reducing agents during probing to minimize non specific interactions Continued on next page Immune Response Biomarker Profiling
83. es on page 11 prior to starting the probing procedure Instructions for blocking the microarray are described below 1 Remove the mailer containing the ProtoArray Control Protein Microarray from storage at 20 C and place immediately at 4 C be sure to use the microarray before the expiration date printed on the box Allow the array to equilibrate in the mailer at 4 C for at least 15 minutes prior to blocking Failure to do so may result in condensation on the array Continued on next page Protein Protein Interaction Probing Procedure Continued Blocking Protocol continued from the previous page Step continued 3 Place one microarray with the barcode facing up into each well of a chilled 4 chamber incubation tray such that the barcoded end of the microarray is near the end of the tray marked with an indented numeral see figure 1a below The indentation in the tray bottom is used as the site for buffer removal see figure 1b arrow 4 Using a sterile pipette add 5 mL Blocking Buffer page 21 equilibrated to 4 C into each chamber with an array Avoid pipetting buffer directly onto the array surface Gently rock the tray to ensure each array is completely immersed in Blocking Buffer Incubate the tray for 1 hour at 4 C on a shaker set at 50 rpm circular shaking After incubation aspirate Blocking Buffer by vacuum or with a pipette Position the tip of the aspirator or pipette into the indentation at
84. eta signal The rabbit anti PKCeta antibody binds to the PKCeta control feature that is spotted twice in each subarray The signals indicate that the antibody is functional and probing is performed properly The signal is also used to check the background 120 Troubleshooting Introduction The table below provides some solutions to possible problems you may encounter when using the ProtoArray Human Protein Microarray for the ASP application Review the expected results section page 120 to verify the probing detection and scanning procedures are performed correctly Weak or no signal Low antibody concentration Perform probing with higher antibody with antibody concentration or increase the incubation time Incorrect probing procedure Follow the recommended protocol for probing Be sure all incubations are performed at 4 C Prepare the Blocking Buffer and Washing Buffer fresh as described on pages 113 Avoid prolonged exposure of detection reagents labeled with fluorescent dye to light Incorrect scanning or Scan the array at suitable wavelength for the imaging detection system used and place the array in the slide holder such that the proteins on the array are facing the laser source Decrease stringency Decrease the number of washes Perform probing and washing in the absence or lower concentration of detergent or salts High background Improper blocking Prepare the Blocking Buffer fresh as described on page 101
85. excess liquid from array surface 7 Proceed immediately to Probing the Array Continued on next page Kinase Substrate Identification Probing Procedure Continued Probing the Array Instructions for probing the microarray are described below 1 Place the ProtoArray Microarray in a 50 mL conical tube with one third of the slide extended outside of the tube see figure below The barcode should be outside the tube face up lt ru If probing two microarrays as outlined in the Recommended Workflow page 41 e Add 1 uL of y P JATP 3 000 Ci mmol 10 uCi pL to 119 uL of kinase 0 1 100 nM in Kinase Buffer see recipe on page 44 to obtain a final y P ATP concentration of 33 nM for one ProtoArray Protein Microarray e Add 1 uL of y P JATP 3 000 Ci mmol 10 pCi pL to 119 uL of Kinase Buffer see recipe on page 44 without kinase Note Once the ATP is added to the kinase use the kinase ATP mixture immediately for probing the array Do not store the prepared kinase ATP mixture on ice for more than 2 minutes prior to use on the array Pipet mixture gently onto the surface of the ProtoArray Protein Microarray within the conical tube without touching the array surface e First Human Microarray add 120 pL Kinase Buffer containing 50 nM your kinase and 33 nM y P ATP Step 2 e Second Human Microarray add 120 pL Kinase Buffer containing 33 nM y P ATP Step 2 with no kinase e For Control Microarrays add 120 p
86. files to generate files that are compatible with your microarray data acquisition software To acquire ProtoArray data from the image you need ProtoArray Prospector Imager 5 0 or higher The latest version of Prospector Imager is included with ProtoArray Prospector for download at www invitrogen com protoarray Microarray data acquisition software such as GenePix Pro Molecular Devices Corporation or ScanArray Software PerkinElmer Inc are suitable for data acquisition Continued on next page 131 Data Acquisition and Analysis Continued ProtoArray Central 132 The ProtoArray Central Portal provides a web based user interface to retrieve ProtoArray Lot Specific information This information GAL file is required for acquiring the array data If the scanner computer is connected to the Internet connect to the portal If the scanner computer is not connected to the internet download the array specific information to portable media as described below and then download the information onto the scanner computer 1 Connect to the portal at www invitrogen com protoarray and then click on the ProtoArray Lot Specific Information link see arrow under BioMarker Discovery Resources BioMarker Discovery Resources lt Information Protein Spot Information 2 The ProtoArray Lot Specific Information page is displayed 3 Enter the array barcode in the Input Barcode Number box Click on the S
87. from Incyte Corporation and Incyte Corporation reserves all other rights under patents owned by Incyte Corporation For information on purchasing a license under patents owned by Incyte Corporation for use of this product for purposes other than research and development please contact Incyte Corporation Corporate Licensing Route 141 and Henry Clay Boulevard Wilmington DE 19880 Phone 302 498 6825 Fax 302 498 2707 This product is the subject of one or more of U S Patent Nos 5 618 676 5 854 018 5 856 123 5 919 651 and foreign equivalents Rights to use this product are limited to research use only Rights are available from Washington Research Foundation to practice under the above referenced patents for any use by contacting Washington Research Foundation 2815 Eastlake Avenue East Suite 300 Seattle Washington 98102 Tel 206 336 5600 Fax 206 336 5615 139 References Bouvier M Menard L Dennis M and Marullo S 1998 Expression and Recovery of Functional G protein coupled Receptors Using Baculovirus Expression Systems Curr Opin Biotechnol 9 522 527 Boyle S N Michaud G A Schweitzer B Predki P F and Koleske A J 2007 A Critical Role for Cortactin Phosphorylation by Abl Family Kinases in PDGF Induced Dorsal Wave Formation Current Biology 17 445 51 Hollister J Grabenhorst E Nimtz M Conradt H and Jarvis D L 2002 Engineering the Protein N glycosylation Pathway in Insect Cells for
88. ftware or equivalent microarray data acquisition software on the computer Open the saved image 16 bit TIFF file from Step 4 page 130 Note If the image is not saved as a 16 bit TIFF file GenePix Pro software is unable to open the file image Acquire data from ProtoArray experiments as follows For ProtoArray Prospector Imager download the GAL files from ProtoArray Central which defines the array grid required by the microarray data acquisition software Load the GAL file into Imager using the Array List button Make adjustments to the blocks as described in the Imager manual Use spots corresponding to the Alignment Control Kinase PKCeta as reference spots to orient the microarray image Scroll through the image to ensure that the grid is in the proper location for each subarray Adjust the subarray grid manually if needed After the grid is adjusted properly and all features are aligned save the Project and analyze the results Imager automatically opens the Analyzer component of ProtoArray Prospector for data analysis and allows you to select the KSI application and specify the experimental conditions Analyzer then performs the data analysis and shows a summary of results see ProtoArray Prospector manual for details For GenePix Pro Software download the GAL files from ProtoArray Central which defines the array grid required by the microarray data acquisition software Analyze the data and save export
89. g Results with ATP Biotin Ubiquitin and purified E1 and E2 ubiquitination enzymes is shown below Control Array probed with Mdm2 purified E1 and E2 ubiquitination enzymes and Streptavidin Alexa Fluor 647 Conjugate Array Image Boxed Area shown in detail rj Alexa Fluor Ab Alexa Fluor Ab e e The following control features can be observed after probing a ProtoArray Protein Microarray e Alexa Fluor Ab signal This is an antibody labeled with Alexa Fluor 647 The fluorescent antibody signals indicate that the array has been properly scanned and are used as reference spots to orient the microarray and help assign spot identities e Mdmo2signal In the presence of ATP Biotin Ubiquitin and purified E1 and E2 ubiquitination enzymes the Mdm2 substrate printed in each subarray is ubiquitinated The signal is used to verify the probing procedure To orient the results obtained from the GAL file and ProtoArray Prospector with the array image position the microarray image such that the barcode is at the Note NA bottom of the image In this orientation the top left corner of the microarray image is Block 1 94 Troubleshooting Introduction The table below provides some solutions to possible problems you might encounter when using the ProtoArray Microarray for the Ubiquitin Ligase profiling application Review the expected results section page 94 to verify the probing detection and scann
90. ge 135 e Blocking Buffer and Washing Buffer see page 113 e 10X Synthtic Block page 135 e Primary antibody diluted in Washing Buffer see page 113 e Appropriate Alexa Fluor 647 conjugated secondary antibody page 135 keep on ice in dark until immediately before use e Ice bucket e Forceps and deionized water e Clean covered 4 chamber incubation tray Greiner Cat no 96077307 chilled on ice e LifterSlip coverslips Thermo Scientific Cat no 25x601 2 4789 e Shaker capable of circular shaking at 50 rpm place the shaker at 4 C e Microarray slide holder and centrifuge equipped with a plate holder Optional The microarray is placed in an incubation tray during the blocking and washing steps To obtain the best results all incubations of the ProtoArray with various solutions are performed in a 4 chamber covered incubation tray Greiner Cat no 96077307 LifterSlip coverslips Thermo Scientific Cat no 25X601 2 4789 hold a small reagent volume to minimize the amount of valuable probe used and prevent evaporation of reagents If you are using any other coverslip be sure the coverslip is able to completely cover the printed area 20 mm x 60 mm of the glass slide and the coverslip is made of non protein binding material Untreated glass coverslips are not recommended Continued on next page 111 Antibody Specificity Profiling Application Probing Procedure Continued Using Your Own Buffers MOM
91. h the slide only by its edges Tap the slide on its side to remove excess fluid but avoid drying of the array Place on a flat surface or benchtop Pipet 120 uL of the small molecule diluted in SMI Assay Buffer page 60 on top of the array without touching the array surface with the pipette tip dropwise Gently rock the slide about 15 30 seconds for solution to spread Using forceps carefully lay the LifterSlip coverslip on the array to cover the printed area without trapping any air bubbles If bubbles are observed gently lift the LifterSlip and slowly lower the slip again Replace slide in the 4 well tray and cover with lid Incubate 90 minutes at 4 C Add 5 mL SMI Assay Buffer incubate without agitation and remove LifterSlip After about a minute or so the LifterSlip should float off of the ProtoArray Human Protein Microarray Once this occurs use the forceps to carefully remove the LifterSlip Discard the slip Alternatively remove the array and LifterSlip from the well and tilt the slide to allow the LifterSlip slip off the surface Replace the array back into the incubation tray to Wash with 5 mL SMI Assay Buffer with gentle agitation for 5 minutes Aspirate SMI Assay Buffer Repeat wash step three times Remove the array from the 4 well tray using forceps Proceed to Drying the Array Continued on next page 63 Small Molecule Interaction Probing Procedure Continued Probing the Array
92. he microarray are printed in 48 subarrays that are equally spaced in vertical and horizontal directions For details on the subarray layout and control spots on the ProtoArray Control Protein Microarray go to the ProtoArray Central Portal at www invitrogen com protoarray Total Subarrays 48 4 columns x 12 rows Subarray Size 4 400 pM x 4 400 uM Subarray Dimensions 22 rows x 22 columns Median Spot Diameter 110 pM Spot Center to Center Spacing 200 pM Distance Between Subarrays 100 pM Replicates per Sample 2 Printing The control proteins are printed on nitrocellulose coated slides in a dust free ProtoArray and temperature and humidity controlled environment to maintain consistent quality of the microarray The arrays are printed using an automated process on an arrayer that is extensively calibrated and tested for printing the ProtoArray Control Protein Microarray Maintaining The ProtoArray Control Protein Microarrays are produced using rigorous Stringent Quality production and quality control procedures with an integrated data Control management system to ensure consistent results with every array and maximize inter and intra lot reproducibility Pre Printing Quality Control Prior to production the arrayer and supporting components are tested and adjusted to production specifications The quality and performance of pins is critical and all pins are extensively tested and calibrated To maintain protein stabili
93. ible to generate your own biotin tagged probe we recommend Ubiquitin Probe using LanthaScreen Biotin Ubiquitin Invitrogen Cat no PV4379 or PV4380 Because the lysine residues are unmodified during the labeling process these labeled ubiquitin reagents are readily incorporated into ubiquitin protein conjugates and poly ubiquitin chains 85 Ubiquitin Ligase Probing Procedure Introduction Experimental Outline Materials Needed Incubation Trays Coverslips 86 Probe the ProtoArray Human Protein Microarray using your ubiquitination enzymes Instructions are included in this section to probe the ProtoArray Human Protein Microarray using buffer recipes provided in this manual see pages 87 88 for buffer recipes Block the ProtoArray Human Protein Microarray Probe with your ubiquitin ligase mixture Perform detection using an appropriate detection system He Nr Dry the array for scanning ProtoArray Human or Control Protein Microarray page 135 e Biotin Ubiquitin Invitrogen Cat no PV4379 or PV4380 e Blocking Buffer and Assay Buffer see pages 87 88 e Ubiquitin ligase mixture in Assay Buffer see next page e Energy Regeneration Solution Boston Biochem Cat no B 10 e Streptavidin Alexa Fluor 647 2 mg mL Invitrogen Cat no S 32357 e Ice bucket e Deionized water e Clean covered 4 chamber incubation tray Greiner Cat no 96077307 chilled on ice e LifterSlip coverslips
94. icroarray For 100 mL Blocking Buffer prepare fresh reagents as follows 1M HEPES pH 7 5 5 mL 5M NaCl 4mL 10 Triton X 100 800 uL 50 Glycerol 50 mL Glutathione Powder 610 mg 10X Synthetic Block 10 mL 2 Adjust pH to 7 5 with NaOH 3 Add 100 pL of 1M DTT 4 Add water to 100 mL Mix well do not vortex and store on ice until use Blocking Buffer without Synthetic Block and DTT may be prepared the day before the assay Store stock at 4 C for no more than 24 hours Washing Buffer final concentration 1X PBS 1X Synthetic Block 0 1 Tween 20 1 Prepare 60 mL of buffer for each microarray For 1 000 mL Washing Buffer prepare fresh reagents as follows 10X PBS 100 mL 10X Synthetic Block 100 mL 10 Tween 20 10 mL Deionized water to 1 000 mL 2 Mix well do not vortex and store on ice until use Continued on next page 113 Antibody Specificity Profiling Application Probing Procedure Continued Before Starting Important 114 Before starting the probing procedure make sure you have all items on hand especially buffers see pages 113 antibodies in Washing Buffer LifterSlip coverslips see page 111 and incubation tray see page 111 Make sure the buffers are cold Store buffers on ice until use Place an incubation tray on ice to chill until use Review Important Guidelines on page 11 prior to starting the probing procedure We strongly recommend that you probe the ProtoArray Human Protein Mic
95. ient Serves as a negative control and signals are used by ProtoArray Prospector software for background and statistical significance calculations Continued on next page ProtoArray Control Protein Microarray Continued Protein Function Control Spots required for KSI and SMI Radioactive Data Analysis Alignment Control Kinase Kinases autophosphorylate and produce signals which are used PKCeta for orientation of the microarray image also serves as a positive control for the radiolabel and assay conditions Control Kinase Substrate A substrate for the Control Kinase MAPK14 p38 alpha used to MAPKAP verify assay conditions The Control Kinase phosphorylates the Control Kinase Substrate Estrogen Receptor Alpha Binds to tritiated estradiol to produce marker signals which are used for orientation of the microarray image for the radiometric small molecule profiling application CAMK2A Calcium calmodulin A human protein kinase that is used as a positive control for the dependent protein kinase II alpha small molecule profiling application Array Control Protein Control Kinase 10 An Array Control Protein is supplied with each ProtoArray Control Protein Microarray for PPI and allows you to verify probing and detection protocols The Array Control Protein is a recombinant yeast calmodulin kinase Cmk1p expressed with a N terminal BioEase V5 tag and purified from
96. ilter the buffer using a 0 45 um filter to remove any particulate material and store on ice until use e After preparing KSI Blocking Buffer immediately return the remaining 30 BSA to 20 C Continued on next page 43 Kinase Substrate Identification Probing Procedure Continued Preparing Kinase Buffer Calculating the Protein Molar Concentration 44 Kinase Buffer final concentration 100 mM MOPS pH 7 2 1 Nonidet P40 NP 40 100 mM NaCl 1 BSA 5mM MgCl 5 mM MnCl 1mM DTT 1 Prepare 120 uL Kinase Buffer with 1 mM DTT for each microarray For 1 mL Kinase Buffer prepare fresh reagents as follows 10 NP 40 100 pL 1M MOPS pH 7 2 100 pL 5M NaCl 20 pL 30 protease free BSA 33 pL 1M MgCl 5 pL 1M MnCl 5 uL 1M DTT 1 pL Deionized water to 1 mL 2 Sterile filter the buffer using a 0 45 um filter to remove any particulate material and store on ice until use 3 Add 33 nM y P ATP at step 2 of Probing Procedure Kinase Buffer without y 33P ATP may be prepared before the assay Store stock at 20 C After preparing the Kinase Buffer with DTT immediately return the remaining Kinase Buffer and 1 M DTT to 20 C To calculate the molar concentration of your protein kinase use the protein concentration and molecular weight of your protein kinase for the calculation using the formula listed below Protein Concentration uM Protein concentration in mg mL x 1 protein molecular weight in grams
97. imental The experimental workflow for IRBP application is described below Workflow Dilute Serum Sample Probe Human Array with Diluted Serum Sample Scan Array and Acquire Array Image Analyze Results Using ProtoArray Prospector 97 Guidelines for Probing the ProtoArray Microarray Human Protein A number of options are available for probing the ProtoArray Human Protein Microarray Microarray with your own buffers and detection reagents as described below Probing Options Review the information below before proceeding with the probing procedure Probing options can be performed individually or in tandem and include e Probing with your serum or plasma probe to detect novel interactions e Probing with only the detection reagent negative control The negative control allows you to determine signals specific to your probe e Probing with different serum or plasma concentrations to determine the optimal amount of sample for your assay Start with an initial sample concentration If the initial signal is strong with low background confirm the initial results with a second array using the same experimental conditions If the initial results indicate weak signal or an unacceptable signal to noise ratio probe a second array with a different serum or plasma concentration 98 Immune Response Biomarker Profiling Probing Procedure Introduction Important Experimental Outline Materials Needed Instructions are
98. in Probe No signal after western detection using an antibody against the protein Poor or no biotinylation for your protein probe Additional biotinylated bands observed 32 The table below provides some solutions to possible problems you might encounter when using the ProtoArray Microarray for the PPI application Review the expected results section page 30 to verify the probing detection and scanning procedures are performed correctly Based on the initial results you may need to optimize the probing and detection protocol by optimizing the probe concentration buffer formulation incubation time or detection reagents Poor or incomplete transfer Monitor the transfer of pre stained protein standard bands to determine the transfer efficiency Increase the exposure time Confirm the presence of the tag by sequence analysis and ensure the tag is cloned in frame Insufficient exposure time Epitope tag not present or cleaved Perform all purification steps at 4 C and use protease inhibitors to prevent proteolytic cleavage of the tag Incorrect buffers used or the biotinylation reaction is not performed correctly Make sure the protein is in a buffer that does not contain any primary amines such as ammonium ions Tris glutathione imidazole or glycine Make sure the biotinylation reaction was performed correctly using the specified molar ratios and at pH 8 0 Check that the calculations and seri
99. in array lot specific information the GAL file from ProtoArray Central Portal Use the lot specific information to acquire and analyze the data to identify potential kinase substrates as described in this section Note To familiarize yourself with the array and subarray layout you may also download a file showing the subarray layout from ProtoArray Central To access the file go to www invitrogen com protoarray and click Online Tools While downloading the lot specific information files ensure that you are downloading files that are associated with your specific barcode on the array Since lot specific information files are updated frequently based on recently available sequence or protein information make sure that you download the latest version of the lot specific information files The GAL GenePix Array List files describe the location and identity of all spots on the Human and Control Microarrays and are used with the microarray data acquisition software to generate files that contain pixel intensity information for feature spot and non features of the slide The GAL files are available for downloading from the ProtoArray Lot Specific Information available on ProtoArray Central see below Note The GAL files are text files that contain the data in a format specified by GenePix Pro Microarray data acquisition software If you are using any other microarray data acquisition software you can use data from the GAL
100. inal saved image Note If the image is scanned at a different dpi set the fixed rectangular area accordingly For example if the image is scanned at 1200 dpi set the fixed rectangular area to 1200 x 3600 pixel to cover the 1 x 3 array area Save the cropped and resized image tiff file with a new name to a suitable location Be sure the barcode is included in the name of the image Download lot specific information from ProtoArray Central see 132 Start Adobe Photoshop on the computer Open the microarray image tiff acquired in Step 4 previous page Perform the following adjustments to the image e Crop a fixed rectangular area 1 x 3 from each image tiff file corresponding to the array If the spots are not aligned vertically rotate the image to correctly align the spots e Invert the data convert the image from white background with black spots to black background with white spots e Resize the image file to 2550 x 7650 pixels constrained proportions Important Do not adjust the image quality such as contrast or level which can compress the dynamic range of the data set and affect data analysis Save the cropped and resized image tiff file with a new name to a suitable location Be sure the barcode is included in the name of the image Proceed to Data Acquisition and Analysis next page Data Acquisition and Analysis Introduction Important GAL File Materials Needed Download the prote
101. information files ensure that you are downloading files that are associated with the specific barcode on your array Since lot specific information files are updated frequently based on recently available sequence or protein information make sure that you download the latest version of the lot specific information files The GAL GenePix Array List files describe the location and identity of all spots on the protein microarray and are used with the microarray data acquisition software to generate files that contain pixel intensity information for all features on the array The GAL files are available for downloading from the ProtoArray Lot Specific Information available on ProtoArray Central see below Note The GAL files are text files that contain the data in a format specified by GenePix Pro Microarray data acquisition software If you are using any other microarray data acquisition software you can use data from the GAL files to generate files that are compatible with your microarray data acquisition software The ProtoArray Central Portal provides a web based user interface to retrieve ProtoArray Lot Specific information This information GAL file is required for acquiring the array data If the scanner computer is connected to the Internet connect to the portal If the scanner computer is not connected to the Internet download the array specific information to portable media as described below and then transfer the inf
102. ing procedures are performed correctly Based on the initial results you may need to optimize the probing and detection protocol by optimizing the probe concentration buffer formulation incubation time or detection reagents Problem Ubiquitination Array Results Weak or no signal Epitope tag not present or Confirm the presence and accessibility of the tag with protein probe not accessible by appropriate assay Poor biotinylation of protein Make sure the small molecule is in a buffer that does not contain any primary amines such as ammonium ions Tris glutathione imidazole or glycine Make sure the biotinylation reaction was performed correctly using the specified molar ratios and at pH 8 0 Check that the calculations and serial dilutions are performed correctly Low probe concentration Perform probing with higher probe concentration or increase the incubation time Incorrect probing procedure Follow the recommended protocols for probing on page 90 Be sure all incubations are performed at 4 C Prepare the Assay Buffer fresh as described on page 88 Do not allow the array to dry during the probing procedure Avoid prolonged exposure of detection reagents labeled with a fluorescent dye to light Incorrect scanning or Scan the array at suitable wavelength for the imaging detection system used and place the array in the slide holder such that the proteins on the array are facing the laser source Decrease str
103. ingency Decrease the number of washes Perform probing and washing in the absence or lower concentration of detergent or salts Continued on next page 95 Troubleshooting Continued Ubiquitination Array Results Improper blocking Prepare the Blocking Buffer fresh as described on page 88 High background Uneven background 96 Improper washing To obtain the best results perform the recommended washing steps Prepare 0 5 SDS solution fresh as described on page 87 Array dried during probing Do not allow the array to dry during probing Array not dried properly Dry the array as described on page 91 before before scanning scanning High probe concentration Decrease the probe concentration or decrease the incubation time Uneven blocking or washing Improper washing During the blocking or washing steps ensure the array is completely immersed in Blocking Buffer or Assay Buffer and use at least 5 mL buffer in the incubation tray to cover the array completely with buffer To obtain the best results perform the recommended washing steps Prepare 0 5 SDS solution fresh as described on page 87 Portions of array have dried Do not allow the array to dry during probing Improper array handling Protein probe not applied properly Always wear gloves and avoid touching the surface of the array with gloved hands or forceps Take care while inserting the array into the Incubation tray to avoid scratching the
104. ion Probing Procedure Continued Using Your Own Buffers Preparing SMI Assay Buffer Preparing Small Molecule Probes 60 Follow the guidelines listed below for buffer preparation to obtain the best results with microarrays The buffer recipes are listed below e Always use ultra pure water to prepare reagents and buffers e You may use non ionic detergents and reducing agents during probing to minimize non specific interactions e If the protein interaction requires certain co factors be sure to include the co factors in the probing buffer during probing e Prepare SMI Assay Buffer fresh prior to use e Use the recipes described below to prepare your own buffers Recommended buffers are listed below for blocking and washing the arrays You can perform array probing using the recommended buffers and then based on your initial results optimize the buffer formulation SMI Assay Buffer final concentration 50 mM Tris HCl pH 7 5 5 mM MgSO 0 1 Tween 20 10X Synthetic Block 1 Prepare 30 mL buffer for each microarray when using Alexa Fluor labeled probes and 50 mL of buffer for each microarray when using biotin labeled probes For 1 000 ml SMI Assay Buffer prepare fresh reagents as follows 1M Tris HCl pH 7 5 50 mL 1MMgSO 5mL 1096 Tween 20 10 mL 10X Synthetic Block 100 mL Deionized water to 1 000 mL 2 Mix well do not vortex and store on ice until use ProtoArray Human Protein Microarray To probe the microa
105. ith only your detection reagent to detect signals resulting due to interactions between the detection reagent and proteins printed on the array e Due to the large variety of probes and detection systems that can be used for probing the ProtoArray Human Protein Microarray it is not possible to have a single probing protocol that is suitable for all probes and detection systems Use the probing procedure from this section as a starting protocol and based on your initial results empirically determine the probing protocol by optimizing the probe concentration buffer formulation incubation time or detection reagents e Optimization of probing protocol can be easily and rapidly achieved using multiple ProtoArray Human Protein Microarrays Continued on next page 61 Small Molecule Interaction Probing Procedure Continued Blocking Step 62 Instructions for blocking the microarray are described below 1 Immediately place the mailer containing the ProtoArray Human Protein Microarray v5 0 at 4 C upon removal from storage at 20 C and equilibrate the mailer at 4 C for at least 15 minutes prior to use 2 Place ProtoArray Human Protein Microarrays with the barcode facing up in the bottom of a 4 chamber incubation tray such that the barcode end of the microarray is near the tray end containing an indented numeral see figure 1a The indent in the tray bottom is used as the site for buffer removal see figure 1b arrow
106. king Prepare the Blocking Buffer fresh as described on page 101 Improper washing To obtain the best results perform the recommended washing steps Prepare the Washing Buffer fresh as described on page 101 Array dried during probing Do not allow the array to dry during probing Array not dried properly Dry the array before scanning before scanning High serum concentration Decrease the serum concentration or decrease the incubation time Antibody cross reactivity Probe a protein array using only the secondary antibody without the serum sample to detect cross reactivity with the antibody only Continued on next page 107 Troubleshooting Continued Uneven background Uneven blocking or During the blocking or washing steps ensure the washing array is completely immersed in blocking solution or Washing Buffer and use 5 mL buffer in the each chamber of the Incubation Tray to cover the array completely with buffer Improper washing To obtain the best results perform the recommended washing steps Prepare the Washing Buffer fresh as described on page 101 Portions of array have dried Do not allow the array to dry during probing Improper array handling Always wear gloves and avoid touching the surface of the array with gloved hands or forceps Take care while inserting or removing the array from the Incubation Tray to avoid scratching the array surface Serum sample or detection Centrifuge the serum sample or det
107. lder Ensure the array is properly placed and is secure in the holder to prevent any damage to the array during centrifugation Dry the array using a table top centrifuge Centrifuge the array at 200 x g for 1 2 minutes at room temperature in the slide holder if using a centrifuge equipped with a plate rotor or 50 mL conical tube if using a swinging bucket rotor Verify that the array is completely dry Ensure the array is properly placed and is secure in the holder to prevent any damage to the array during centrifugation Place the array in an X ray film cassette Cover the array with a single layer of clear plastic wrap You can check for radioactivity on the array using a Geiger counter Overlay the array with an X ray film or a phosphor screen at least 50 uM resolution Be sure the phosphor screen was erased prior to exposure Expose the arrays for 3 hours Proceed to Image Acquisition and Processing next page Image Acquisition and Processing Introduction Materials Needed Scanning the Array Data Acquisition and Analysis Once you have exposed the ProtoArray Microarray to X ray film or phosphor screen scan the film or phosphor screen to acquire a TIFF image that is required for microarray data analysis To make the image compatible with the microarray data acquisition software process the image using ProtoArray Prospector Imager or Adobe Photoshop image analysis software as described on page 130 Imagi
108. lly or in tandem and include e Probing with MAPK14 p38 alpha positive control The result from the positive control helps to determine signals specific to your probe e Probing with your kinase of interest with y P A TP to help you determine background signal and possible array surface interactions The recommended protein kinase concentration for probing the ProtoArray Human Protein Microarray is 50 nM A number of options are available for probing the human microarray with the protein kinase of interest using pre made reagents from ProtoArray Human Protein Microarray v5 0 KSI kit or your own buffers and detection reagents as described below Review the information below before proceeding with the probing procedure Probing options can be performed individually or in tandem and include e Probing with your kinase of interest at 50 nM with y P ATP to identify potential substrates e Probing with only the buffer and no kinase negative control in the presence of radiolabeled y P ATP The negative control allows you to determine signals specific to your probe e Probing with MAPK14p38 alpha positive control The result from the positive control helps to determine which signals are specific to your kinase e Probing with different probe concentrations to determine the optimal amount of probe for your assay Start with an initial probe concentration If the initial signal is strong with low background confirm th
109. lutathione imidazole or glycine Make sure the biotinylation reaction was performed correctly using the specified molar ratios and at pH 8 0 Check that the calculations and serial dilutions are performed correctly Low probe concentration Perform probing with higher probe concentration or increase the incubation time Incorrect probing procedure Follow the recommended protocols for probing on pages 63 and 64 Be sure all incubations are performed at 4 C Prepare the SMI Assay Buffer fresh as described on page 60 Do not allow the array to dry during the probing procedure Avoid prolonged exposure of detection reagents labeled with a fluorescent dye to light Incorrect scanning or Scan the array at suitable wavelength for the imaging detection system used and place the array in the slide holder such that the proteins on the array are facing the laser source Decrease stringency Decrease the number of washes Perform probing and washing in the absence or lower concentration of detergent or salts Continued on next page 69 Troubleshooting Continued SMI Array Results Improper blocking Prepare the SMI Assay Buffer fresh as described on page 60 High background Uneven background 70 Improper washing To obtain the best results perform the recommended washing steps Prepare the SMI Assay Buffer fresh as described on page 60 Array dried during probing Do not allow the array to dry during probing
110. massie is a registered trademark of Imperial Chemical Industries PLC Cy5 is a trademark of Amersham Biosciences GenePix is a registered trademark of Molecular Devices Corporation GenTel is a registered trademark of GenTel Biosciences Inc LifterSlip is a trademark of Thermo Scientific Microsoft is a registered trademark of Microsoft Corp ScanArray is a registered trademark of PerkinElmer Inc 140 invitrogen Corporate Headquarters Invitrogen Corporation 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
111. materials Monitor work area continuously for radiation contamination Dispose of radioactive waste properly After you have completed your experiments monitor all work areas equipment and yourself for radiation contamination Follow all the radiation safety rules and guidelines mandated by your institution Any material in contact with a radioactive isotope must be disposed of properly This includes any reagents that are discarded during the probing procedure e g washes Contact your safety department for regulations regarding radioactive waste disposal Guidelines for Probing the ProtoArray Microarray Introduction Control Protein Microarray Probing Options Human Protein Microarray Probing Options Instructions are included in this section for probing a ProtoArray Protein Microarray using your protein kinase or the Control Kinase and radiolabeled ATP Various options are available for performing the probing procedure see below Experimental workflows for probing are shown on pages 36 37 If you are a first time user of the ProtoArray Human Protein Microarray we recommend that you probe a ProtoArray Control Protein Microarray available from Invitrogen page 135 prior to probing the human microarray The ProtoArray Control Protein Microarray contains various controls and protein interactors printed on the array to allow you to validate probing and detection protocols Probing options can be performed individua
112. ments with additional arrays to ensure e Reproducibility Probe protein arrays using a similar or a different probe concentration to observe similar interactions e Specificity Probe protein arrays with the detection reagent used to visualize the interactions and also different antibodies to identify interactions specific to your antibody of interest and also identify any non specific interactions There are several additional appropriate assay formats for validation of antibody protein interactions including ELISA Luminex and immunoblotting Continued on next page 119 Expected Results for Antibody Specificity Profiling Applicaton Control Array Results obtained after probing the ProtoArray Control Protein Microarray v5 0 Probing Results with a rabbit anti PKCeta antibody followed by detection with Alexa Fluor 647 labeled secondary antibody is shown below Control Array with rabbit anti PKCeta antibody and Alexa Fluor 647 anti rabbit IgG Array Image Boxed Area shown in detail PKCeta Alexa Fluor Ab E Alexa PKCeta Fluor Ab ee ee The following control features can be observed after probing a ProtoArray Protein Microarray e Alexa Fluor Ab signal This is an antibody labeled with Alexa Fluor 647 The fluorescent antibody signals indicate that the array has been properly scanned and are used as reference spots to orient the microarray and help assign spot identities e PKC
113. microarrays The buffer recipes are listed below e Always use ultra pure water to prepare reagents and buffers e You may use non ionic detergents and reducing agents during probing to minimize non specific interactions e If the protein interaction requires certain co factors be sure to include the co factors in the probing buffer during probing e Prepare Tritium SMI Assay Buffer fresh prior to use Freshly prepared buffers are best for blocking slides Do not store Tritium SMI Assay Buffer for more than 24hrs e Use the recipes described below to prepare your own buffers Recommended buffers are listed below for blocking and washing the arrays You can perform array probing using the recommended buffers and then based on your initial results optimize the buffer formulation Tritium SMI Assay Buffer final concentration 50 mM Tris HCl pH 7 5 5 mM MgSO 0 1 Tween 20 100 mM NaCl Optional 1 BSA or Casein Optional 2 Prepare 125 ml buffer for each microarray If using the optional Tritium SMI Assay Buffer with NaCl prepare an additional 40 ml of buffer without NaCl for each microarray For 1 000 ml Tritium SMI Assay Buffer prepare fresh reagents as follows 1M Tris HCl pH 7 5 50 mL 1 M MgSO 5mL 10 Tween 20 10 mL 5 M NaCl Optional 20 mL 30 protease free BSA Optional 33 4 mL OR Casein Hammarsten Grade Optional 10g Deionized water to 1 000 mL 3 Mix well do not vortex and store on ice until use Ass
114. n page 126 Expression and Almost all clones used to generate the human protein collection are entry clones Purification of consisting of a human ORF cloned into a Gateway entry vector Each entry Human Proteins clone is subjected to an LR recombination reaction with a Gateway destination vector to generate an expression clone The expression clone is then used to express the protein as an N terminus GST fusion protein in some clones using the Bac to Bac Baculovirus Expression System available from Invitrogen For more information on the Bac to Bac Baculovirus Expression System visit www invitrogen com The LR reaction mix obtained after performing the LR reaction is transformed into competent DH10Bac E coli to generate a recombinant bacmid The high molecular weight recombinant bacmid DNA is isolated and transfected into Sf9 insect cells to generate a recombinant baculovirus that is used for preliminary expression experiments After the baculoviral stock is amplified the high titer stock is used to infect Sf9 insect cells for expression of the recombinant protein of interest The expressed proteins are purified by affinity chromatography under high throughput conditions optimized to obtain maximal protein integrity function and activity Following purification each protein is assayed for purity and expected molecular weight Continued on next page ProtoArray Human Protein Microarray Continued Printing the The pu
115. n see page vi for efficient in vitro biotinylation of your protein of interest The kit includes reagents and buffers for in vitro biotinylation and removal of free biotin The FluoReporter Biotin Quantitation Assay Kit see page vi can be used to assess the number of biotin labels on the protein Continued on next page Preparing the Protein Probe Continued Protein Amount and Quality Purify the protein using native conditions Proteins should be gt 90 pure as determined by Coomassie staining Check the presence of the tag using western detection or ELISA Note To ensure that the tag is accessible under native conditions used for probing microarrays perform ELISA of your protein probe with the tag Check the functionality of the protein probe using a method of choice Make sure the protein probe is soluble and active in buffers used for probing the microarray The recommended protein concentration range for probing each human protein microarray is 100 nM 10 uM for biotinylated proteins and 10 nM 1 uM for V5 tagged proteins If you are using in vitro biotinylated proteins for probing Resuspend the purified protein probe in a buffer 50 mM that does not contain any primary amines such as ammonium ions Tris glutathione imidazole or glycine If the buffer contains primary amines sufficiently dialyze the protein probe against 50 mM HEPES buffer pH 7 4 containing 100 mM NaCl or PBS Determine the approximate molec
116. n Protein Microarrays we have typically observed a true positive rate of 80 for serine threonine protein kinases A true positive signal is defined as a phosphorylation signal observed on a protein microarray that is validated as a substrate using an in vitro solution assay The kinase substrate identification assay depends on various factors such as the buffer composition kinase activity concentration assay conditions ATP concentration protein sequence and the amount of protein on the array It is possible that some proteins reported in literature as substrates for the kinase may not be identified as kinase substrates on the array When comparing the kinase substrate data obtained from ProtoArray experiments to kinase annotated substrates as reported in the literature it is important to review the experimental conditions used for identifying a protein as a substrate for the kinase In many cases proteins annotated in the literature as kinase substrates have been identified using in vivo based approaches which are not always conclusive Sometimes the identified signals on the array may be due to the interaction of an array protein with radiolabeled ATP or autophosphorylated protein kinase thereby causing false positive results To minimize the number of false positive signals arising due to non specific interaction and to decrease the number of signals not arising from protein kinase phosphorylation of array proteins wash the kinase tr
117. n a slide holder or a sterile 50 mL conical tube if you do not have a slide holder Ensure the array is properly placed and is secure in the holder to prevent any damage to the array during centrifugation Briefly dip the slide holder containing the arrays into room temperature distilled water three times to remove salts If you are not using a slide holder dip the array into a 50 mL conical tube filled with room temperature distilled water three times Centrifuge the array in the slide holder or 50 mL conical tube at 200 x g for 1 minute in a centrifuge equipped with a plate rotor if you are using the slide holder at room temperature Verify the array is completely dry After drying store the arrays vertically or horizontally in a slide box protected from light Avoid prolonged exposure to light To obtain the best results scan the array within 24 hours of probing Proceed to Scanning and Data Analysis next page Scanning and Data Analysis Introduction Materials Needed Scanning the Array Data Acquisition and Analysis ProtoArray Prospector Results Once you have probed the ProtoArray with your serum or plasma sample scan the microarray using a suitable microarray scanner Instructions are included in this section to scan the microarray using a fluorescence microarray scanner Imaging hardware A suitable scanner is required to scan the ProtoArray Microarray The scanner specifications are listed page 123 For
118. n anti human IgG is used for detection The Human IgG signals are used to verify proper probing and detection reagents e Anti human IgG Signal A protein gradient of goat anti human IgG is printed on each subarray The IgG from human serum binds to the anti human IgG on the array and is used to verify proper probing and detection reagents 106 Troubleshooting Introduction The table below provides some solutions to possible problems you may encounter when using the ProtoArray Human Protein Microarray for IRBP Review the expected results section page 106 to verify the probing detection and scanning procedures are performed correctly Weak or no signal Low serum concentration Perform probing with higher serum with serum sample concentration or increase the incubation time Incorrect probing procedure Follow the recommended protocol for probing Be sure all incubations are performed at 4 C Prepare the Blocking Buffer and Washing Buffer fresh as described on page 101 Avoid prolonged exposure of detection reagents labeled with fluorescent dye to light Incorrect scanning or Scan the array at suitable wavelength for the imaging detection system used and place the array in the slide holder such that the proteins on the array are facing the laser source Decrease stringency Decrease the number of washes Perform probing and washing in the absence or lower concentration of detergent or salts High background Improper bloc
119. n is influenced by the affinity of the tritium labeled small molecule for its protein target Generally the tritium labeled ligand should be probed at the highest achievable concentration due to the limited sensitivity of detection of the tritium signal Gently place a coverslip over the surface of the ProtoArray Protein Microarray using forceps taking care to avoid capturing bubbles Position the ProtoArray Protein Microarray with coverslip within the conical tube with the printed side of the array facing up Cap the tube Place the tube on a flat surface such that the printed side of the array is facing up and the tube is as level as possible If needed tape the conical tube on the flat surface to avoid any accidental disturbances Incubate the array at 4 C for 90 minutes without shaking Remove conical tube containing ProtoArray Protein Microarrays from incubator and add 40 mL Tritium SMI Assay Buffer to the tube Incubate the array in buffer for 30 seconds at room temperature The glass coverslip will float off Do not remove the coverslip with forceps if it is not dislodged from the array Using forceps carefully remove the dislodged coverslip without touching the array surface Discard the coverslip appropriately as radioactive waste Decant the Tritium SMI Assay Buffer Be sure to dispose of the radioactive waste properly Add 40 mL of fresh Tritium SMI Assay Buffer to the tube Incubate the array for 30 seconds at room tem
120. nalyze data with ProtoArray Prospector using the guidelines on page 128 to determine significant signals with the controls and your protein probe Continued on next page Scanning and Data Analysis Continued Analyzing ProtoArray Prospector Results The Next Step After data analysis ProtoArray Prospector presents a summary of the analyzed data in a table format see ProtoArray Prospector manual for details The proteins that score as positive in the experiment are proteins that satisfy the basic program options Review the information on page 94 Expected Results to help with data interpretation We recommend validating the interactions as described below After identifying potential ubiquitin ligase substrates on the ProtoArray Human Microarray you may reproduce the result using The ProtoArray Technology with additional arrays to ensure e Reproducibility Probe the human array using a similar or a different concentration of ubiquitination enzymes to address reproducibility e Specificity Probe a human array with a different ubiquitin ligase or in the absence of the E3 ligase to identify substrates specific to your ubiquitin ligase of interest OR e Perform solution based assays to assess ubiquitination of candidate substrates in vitro Continued on next page 93 Expected Results for Ubiquitin Ligase Control Array Results obtained after probing the ProtoArray Control Protein Microarray v5 0 Probin
121. nce of free biotin from the labeling process The recommended protein probe concentration range for probing the ProtoArray Human Protein Microarray is 100 nM 10 uM for biotinylated proteins and 10 nM 1 uM for V5 tagged proteins A number of options are available for probing the ProtoArray Human Protein Microarray with the protein probe of interest using pre made reagents from ProtoArray Human Protein Microarray v5 0 PPI kits or your own buffers and detection reagents as described below Review the information below before proceeding with the probing procedure Probing options can be performed individually or in tandem and include e Probing with your protein probe to detect novel interactions e Probing with only the detection reagent negative control The negative control allows you to determine signals specific to your probe e Probing with the Array Control Protein supplied with the ProtoArray Control Protein Microarray positive control The result from the positive control helps to determine signals specific to your probe e Probing with different probe concentrations to determine the optimal amount of probe for your assay Start with an initial probe concentration If the initial signal is strong with low background confirm the initial results with a second array using the same experimental conditions If the initial results indicate weak signal or an unacceptable signal to noise ratio probe a second array with a diffe
122. ng hardware A standard desktop flatbed image scanner that provides at least 50 uM resolution gt 600 dpi to scan the X ray film OR A phosphorimager that provides at least 50 uM resolution to acquire the image from a phosphor screen see page 129 for phosphorimagers that have been tested with ProtoArray Microarrays Data acquisition software We recommended GenePix Pro v6 or later Molecular Devices Corporation or ScanArray Acquisition Software PerkinElmer Inc as microarray data acquisition software for analysis of images For detailed instructions on scanning the microarray refer to Image Acquisition and Processing for Radioactive Assays page 129 1 Develop the X ray film or process the phosphor screen according to the manufacturer s recommendations 2 Scan the X ray film on a standard scanner or scan the phosphor screen on a phosphorimager to generate a 16 bit TIFF image file 3 Process the image using ProtoArray Prospector Imager 4 Save the adjusted microarray image For detailed instructions on Data Acquisition and Analysis refer to page 131 1 Acquire an image tiff from the phosphor screen 2 Use the barcode information on the array to download the GAL file from ProtoArray Central as described on page 132 3 Use the GAL file and ProtoArray Prospector to acquire pixel intensity values for all features on the array and analyze data to determine significant signals Continued on next page
123. ng in vitro biotinylated protein probes for detection we recommend that you also probe a Control Microarray with your biotinylated probe to validate the biotinylation of your protein probe and determine background levels Details on the ProtoArray Control Protein Microarray are described in this section Do not use the ProtoArray Control Protein Microarray for detecting novel protein interactions with your probe of interest The Control Microarray contains only a defined set of control proteins and does not contain the entire set of protein content printed on the ProtoArray Human Protein Microarray The ProtoArray Control Protein Microarray specifications are listed below Dimensions 1 inch x 3 inch 25 mm x 75 mm Material Glass slide coated with a thin film of nitrocellulose The nitrocellulose coated slide is from GenTel BioSciences Inc Thin film nitrocellulose slides are manufactured by Gentel Biosciences Inc using a proprietary surface chemistry owned by Decision Biomarkers Inc Thin film nitrocellulose slides are covered by US Patent 6 861 251 7 297 497 and 7 384 742 Each microarray has a barcode for tracking samples The barcode number is also used to retrieve array specific information from the portal page 126 Continued on next page ProtoArray Control Protein Microarray Continued Control Array The ProtoArray Control Protein Microarray specifications are listed below Specifications The proteins on t
124. ntle agitation Remove Washing Buffer by aspiration see Blocking Step Repeat wash step 4 more times Add 5 mL of Alexa Fluor 647 conjugated secondary antibody diluted in Washing Buffer if necessary Note This step is not needed if performing detection using a labeled primary antibody or Streptavidin Alexa Fluor 647 Conjugate Incubate for 90 minutes at 4 C with gentle circular shaking 50 rpm Remove secondary antibody by aspiration see Blocking Step Wash with 5 mL fresh Washing Buffer for 5 minutes with gentle agitation Remove Washing Buffer by aspiration see Blocking Step Repeat wash step 4 more times Proceed to Drying the Array below Remove the array from the 4 chamber incubation tray see page 26 Step 1 Place the array in a slide holder or a sterile 50 mL conical tube if you do not have a slide holder Ensure the array is properly placed and is secure in the holder to prevent any damage to the array during centrifugation Briefly dip the slide holder containing the arrays into room temperature distilled water three times to remove salts If you are not using a slide holder dip the array into a 50 mL conical tube filled with room temperature distilled water three times Dry the ProtoArray Microarray by centrifugation Centrifuge the array at 200 x g for 1 2 minutes at room temperature in the slide holder if using a centrifuge equipped with a plate rotor or 50 mL conical tube if using a swinging bucket r
125. numeral end and gently prying the array upward see figure previous page Using a gloved hand pick up the microarray by holding the array by its edges 2 Place the array in a slide holder or a sterile 50 mL conical tube if you do not have a slide holder Ensure the array is properly placed and is secure in the holder to prevent any damage to the array during centrifugation Briefly dip the slide holder containing the arrays into room temperature distilled water three times to remove salts If you are not using a slide holder dip the array into a 50 mL conical tube filled with room temperature distilled water three times 3 Centrifuge the array in the slide holder or 50 mL conical tube at 200 x g for 1 minute in a centrifuge equipped with a plate rotor if you are using the slide holder at room temperature Verify the array is completely dry After slides have been probed and dried they can be stored either vertically or horizontally 4 After drying store the arrays vertically or horizontally in a slide box protected from light Avoid prolonged exposure to light as it will diminish signal intensities To obtain the best results scan the array within 24 hours of probing 5 Proceed to Scanning and Data Analysis next page Continued on next page 91 Scanning and Data Analysis Introduction Materials Needed Scanning the Array Data Acquisition and Analysis 92 Once you have probed the ProtoArray with your ligase p
126. oarray Microarray data acquisition software such as GenePix Pro Molecular Devices Corporation or ScanArray Software PerkinElmer Inc are also suitable for data acquisition 4 Develop the X ray film or process the phosphor screen according to the manufacturer s recommendations 5 Scanthe X ray film on a standard scanner or scan the phosphor screen on a phosphorimager to generate a 16 bit TIFF image file 6 Process the image using ProtoArray Prospector Imager 7 Save the adjusted microarray image After exposing the X ray film or phosphor screen to the ProtoArray Microarray scan the film or phosphor screen to obtain a 16 bit TIFF image file that is required for microarray data analysis Brief scanning guidelines are described below For details refer to the manufacturer s recommendations on using the scanner or phosphorimager 1 Remove the X ray film or phosphor screen from the cassette Keep the array covered in clear plastic wrap in the dark for use later if a longer exposure time is needed Develop the X ray film 3 Scanthe X ray film using a standard scanner or scan the phosphor screen using a phosphorimager to obtain a 16 bit TIFF file Include the barcode in the area for maintaining a record and scan the array to provide a high resolution image 50 pM 4 Savethe image file to a suitable location Continued on next page 129 Image Acquisition and Processing for Radioactive Assays Continued Image
127. obing Procedure Continued Probing the Array 116 1 Pipette 120 pL of primary antibody diluted in Washing Buffer page 113 on top of the array without touching the array surface with the pipette tip The liquid quickly spreads over the nitrocellulose coating Carefully lay the LifterSlip on the array to cover the printed area without trapping any air bubbles The white raised edges of the lifter slip should face the array Gently adjust the LifterSlip to remove any air bubbles Do not allow any part of the array surface to dry before adding the next solution as it will cause high and or uneven background Incubate the array in the tube for 90 minutes at 4 C without shaking Wash with 5 mL Washing Buffer for 5 minutes using gentle agitation 50 rpm Carefully remove the LifterSlip with forceps without touching the array surface Discard the LifterSlip Remove Washing Buffer by aspiration see Blocking Step Repeat wash steps 4 more times Add 5 mL secondary antibody diluted in Washing Buffer to the indentation at the numbered end of the incubation tray and allow the liquid to flow across the slide surface To avoid local variations in fluorescence intensity and background avoid direct contact with the array Do not pour the antibody solution directly on the slide Incubate the array in the tube for 90 minutes at 4 C with gentle circular shaking 50 rpm Remove secondary antibody by aspiration See Blocking Step
128. olution fresh as described on page 43 Portions of array have Do not allow the array to dry during probing dried Improper array handling Always wear gloves and avoid touching the surface of the array with gloved hands or forceps Take care while inserting the array into the tube to avoid scratching the array surface Radiolabeled ATP or Centrifuge the y P ATP or buffer to remove buffer contains precipitates precipitates prior to probing the array Poor spot resolution Incorrect scanner or Be sure the scanner or phosphorimager is capable of phosphorimager used providing at least 50 uM resolution Improper handling of Be sure to allow the mailers with arrays to arrays equilibrate at 4 C for at least 15 minutes prior to use Improper covering of Properly cover the array with a single layer of clear arrays plastic wrap without any creases Signals from It is normal for signals from duplicate spots to merge duplicate spots are sometimes The merging of spots does not affect data merged analysis 55 Small Molecule Identification SMI Fluorescent Application Experimental Overview Experimental The recommended experimental steps for SMI application with Alexa Fluor Steps 647 labeled or biotinylated small molecules are outlined below Step Action Page no 1 Block ProtoArray Human Protein Microarray with 5 mL 62 SMI Assay Buffer 2 Probe ProtoArray Human Protein Microarray with 120 pL 63 of small molecule in SMI
129. on below before proceeding with the probing procedure Probing options can be performed individually or in tandem and include e Probing with your antibody probe to detect novel interactions e Probing with only the detection reagent negative control The negative control allows you to determine signals specific to your probe e Probing with different antibody concentrations to determine the optimal amount of antibody for your assay Start with an initial antibody concentration If the initial signal is strong with low background confirm the initial results with a second array using the same experimental conditions If the initial results indicate weak signal or an unacceptable signal to noise ratio probe a second array with a different antibody concentration 110 Antibody Specificity Profiling Application Probing Procedure Introduction Experimental Outline Materials Needed Incubation Trays Coverslips Instructions are included in this section to probe the ProtoArray Human Protein Microarray using an unlabeled primary antibody followed by an Alexa Fluor 647 labeled secondary antibody If you are preparing your own buffers see page 113 for buffer recipes 1 Block the ProtoArray Human Protein Microarray 2 Probe with your primary antibody 3 Perform detection using Alexa Fluor 647 labeled secondary antibody 4 Dry the array for scanning e ProtoArray Human or Control Protein Microarray v5 0 pa
130. ontrol Protein and Anti V5 Alexa Fluor 647 Antibody Array Image Boxed Area shown in detail Array image Boxed Area shown in detail Biotin Ab IEEE Alexa Fluor Ab Calmodulin Alexa V5 Control Protein Fluor ease T A FT Feature is Cmd1p or CALM2 depending on the array lot Refer to the lot specific GAL file for the specific identity of the protein Calmodulin Alexa V5 Control Protein Fluor eooee ET Feature is Cmd1p or CALM2 depending on the array lot Refer to the lot specific GAL file for the specific identity of the protein List of control features continued from the previous page e Calmodulin signal The Array Control Protein BioEase V5 calmodulin kinase binds to the calmodulin Cmd1p or CALM2 printed on the array The signal is used to verify the probing procedure Refer to the lot specific GAL file for the specific identity of the protein Note The Array Control Protein contains an N terminal BioEase and V5 epitope tag The BioEase in vivo biotinylation of the protein during expression TM tag facilitates To orient the results obtained from the GAL file and ProtoArray Prospector with Note the array image position the microarray image such that the barcode is at the bottom of the image In this orientation the top left corner of the microarray image is Block 1 31 Troubleshooting Introduction Problem Prote
131. ontrol Microarray to verify probing and detection protocols Dry the microarray 91 Scan slide with fluorescence microarray scanner 92 Download the protein array lot specific information the 92 GAL file from ProtoArray Central Portal to acquire and analyze the data using ProtoArray Prospector to identify potential substrates Continued on next page 84 Guidelines for Probing the ProtoArray Microarray Human Protein A number of options are available for probing the ProtoArray Human Protein Microarray Microarray with your own buffers and detection reagents as described below Probing Options Review the information below before proceeding with the probing procedure Probing options can be performed individually or in tandem and include e Probing with your ubiquitination enzymes to detect novel interactions e Probing with only the detection reagent negative control The negative control allows you to determine signals specific to your probe e Probing with different probe concentrations to determine the optimal amount of probe for your assay Start with an initial probe concentration If the initial signal is strong with low background confirm the initial results with a second array using the same experimental conditions If the initial results indicate weak signal or an unacceptable signal to noise ratio probe a second array with a different probe concentration Biotin Tagged While it is poss
132. ormation onto the scanner computer 1 Connect to the portal at www invitrogen com protoarray and then click on the ProtoArray Lot Specific Information link see arrow under BioMarker Discovery Resources BioMarker Discovery Resources Products Protocols amp Applications Resources Online Tools ProtoArray Lot Specific E Information Protein Spot Information 2 The ProtoArray Lot Specific Information page is displayed Continued on next page Data Acquisition and Analysis Continued ProtoArray Protocol continued from previous page Central continued 3 Enter the array barcode in the Input Barcode Number box see arrow Click on the Search button Input Barcode Number s Search For each input barcode the following files are displayed GAL file LotNumber gal This file is essential for data acquisition by the software and defines spot locations and identities of all protein spots on the array The file also includes the detected protein concentration information in relative fluorescent units for each spot Protein Information File LotNumber info txt This file contains a listing and description of human proteins on the array Protein Sequence File LotNumber seq txt This tab delimited text file lists the GenBank accession number Ultimate ORF Clone ID number if available FASTA header and amino acid sequence of the ORF for each array protein Control Data File LotNumber_control txt
133. otein Microarray v5 0 if you are using the recommended workflow for probing the array y P ATP 3 000 Ci mmol 10 uCi uL 0 5 SDS KSI Blocking Buffer and Kinase Buffer page 43 44 for recipes 0 45 um filters Millipore SLHVR25LS Clean covered 4 chamber incubation tray Greiner Cat no 96077307 chilled on ice Protein Kinase supplied by the user in Kinase Buffer page 44 Incubator set to 30 C Sterile 50 mL conical tubes Coverslips VWR Cat no 48404 454 Ice bucket Deionized water Shaker X ray film or phosphor screen provide at least 50 uM resolution and instrumentation to acquire the image provide at least 50 um resolution X ray film cassette Clear plastic wrap Microarray slide holder and centrifuge equipped with a plate holder Optional You will need coverslips that are able to completely cover the printed area 20 mm x 60 mm of the glass slide and hold a small reagent volume to minimize the amount of valuable probe used and prevent evaporation of reagents We recommend using glass coverslips VWR Cat no 48404 454 If you are preparing your own buffers follow the guidelines listed below for buffer preparation to obtain the best results with microarrays The buffer recipes are listed on the next page Always use ultra pure water to prepare reagents and buffers You may use non ionic detergents and reducing agents during probing to minimize non specific interactions If the kinase assay requires cer
134. otor Verify that the array is completely dry After drying store the arrays vertically or horizontally in a slide box protected from light Avoid prolonged exposure to light To obtain the best results scan the array within 24 hours of probing Proceed to Scanning and Data Analysis next page 27 Scanning and Data Analysis Introduction Materials Needed Scanning the Array Data Acquisition and Analysis 28 Once you have probed the ProtoArray with your protein probe scan the microarray using a suitable microarray scanner After scanning and saving an image of the array download the protein array lot specific information from the ProtoArray Central Portal Use the lot specific information to acquire and analyze the data to identify protein protein interactions Imaging hardware A suitable scanner is required to scan the ProtoArray Microarray The scanner specifications are listed page 123 For a list of scanners to use with ProtoArray Microarrays see page 124 Data acquisition software We recommended GenePix Pro v6 or later Molecular Devices Corporation or ScanArray Acquisition Software PerkinElmer Inc as microarray data acquisition software for analysis of images For detailed instructions on scanning the microarray refer to Scanning Arrays Using a Fluorescence Scanner page 123 Insert array into the fluorescence microarray scanner Adjust scanner settings Preview the microarray and adjust
135. ouching the surface of the array with gloved hands or forceps Take care while inserting the array into the Incubation tray to avoid scratching the array surface Protein probe not applied Apply the probe solution and LifterSlip or properly equivalent coverslip to the array as described in the manual To avoid drying of the array surface make sure the coverslip covers the printed area of the array and adjust the coverslip if needed Probe or detection reagents Centrifuge the probe or detection reagents to contain precipitates remove precipitates prior to probing the array 34 Kinase Substrate Identification KSI Application Experimental Overview Experimental The recommended experimental steps for KSI application are outlined below Steps Detailed experimental workflow is shown on the next page Step Action Page no 1 Purify your protein kinase of interest using a method of 40 choice or purchase the protein kinase of interest from Invitrogen 2 Block ProtoArray Human Protein Microarray with 5 mL 46 KSI Blocking Buffer 3 Probe the ProtoArray Human Protein Microarray with the 47 protein kinase of interest in the presence of radiolabeled ATP Optional If you are a first time user of the ProtoArray Human Protein Microarray perform a control probing using a ProtoArray Control Protein Microarray to verify the assay protocol Dry the microarray 48 Expose the microarray to X ray film or phospho
136. perature Decant buffer Repeat wash step one more time Be sure to dispose of the radioactive waste properly If Tritium SMI Assay Buffer with NaCl is used complete one additional wash with Tritium SMI buffer lacking NaCl Proceed to Drying the Array Continued on next page 77 Tritium Radiolabeled Small Molecule Interaction Probing Procedure Continued Drying the Array 78 1 Remove the array from the chamber at the end of the probing procedure Tap one edge of the array gently on a laboratory wipe for a few seconds to drain any buffer Place each array in a slide holder or a sterile 50 mL conical tube if you do not have a slide holder Ensure the array is properly placed and is secure in the holder to prevent any damage to the array during centrifugation Centrifuge the array in the slide holder or 50 mL conical tube at 200 x g for 1 minute in a centrifuge equipped with a plate rotor if you are using the slide holder at room temperature Verify the array is completely dry Using transparent tape adhere the slides to an 8X10 Exeter Conservation Board or thick filter paper of similar size Only tape the top and bottom edges of the slide without covering any array area The adhesion helps to prevent unwanted movement during the long exposure time and also helps to prevent the tritium from transferring on to the screen Place ProtoArray Protein Microarrays in X ray film cassette and directly overlay with a tritium
137. pletely with buffer Improper washing To obtain the best results perform the recommended washing steps Prepare Tritium SMI Assay Buffer fresh as described on page 74 Portions of array have Do not allow the array to dry during probing dried Improper array handling Always wear gloves and avoid touching the surface of the array with gloved hands or forceps Take care while inserting the array into the tube to avoid scratching the array surface Reagents or buffer contains Centrifuge the reagents or buffer to remove precipitates precipitates prior to probing the array Signals from It is normal for signals from duplicate spots to merge duplicate spots are sometimes The merging of spots does not affect data merged analysis 83 Ubiquitin Ligase Profiling Application Experimental Overview Experimental The recommended experimental steps for probing ProtoArray Human Protein Steps Microarray with a ubiquitin ligase to identify interactors and or substrates of E2 E3 ubiquitin ligase modifying enzymes are outlined below Step Action Page no 1 Block ProtoArray Human Protein Microarray with 5 mL 90 Blocking Buffer 2 Prepare ubiquitin ligase mixture s and incubate at 30 C for 87 5 minutes 3 Probe ProtoArray Human Protein Microarray with 100 uL 91 ubiquitin ligase mixture Optional If you are a first time user of the ProtoArray Human Protein Microarray perform a control probing using a ProtoArray C
138. precipitates prior to probing precipitates the array 122 Scanning Arrays Using a Fluorescence Scanner Introduction Non Fluorescent Scanners Materials Needed Experimental Outline Scanner Specifications Once the ProtoArray Microarray has been probed the array is scanned to aquire fluorescent signal In this section guidelines are provided for selecting a suitable fluorescence microarray scanner and instructions are given on scanning the microarray for the PPI SMI Fluorescent IRBP ASP and Ubiquitin Ligase profiling applications If you have used a non fluorescent detection system such as chemiluminescence or radioactivity an imaging system with a CCD camera such as the Alphaimager Imaging System for chemiluminescence detection or a phosphorimager scanner such as the PerkinElmer Cyclone phosphor imaging system for detecting radioactivity is required to capture the signal Follow the manufacturer s recommendations to scan the microarray A suitable fluorescence microarray scanner is needed to scan the ProtoArray Microarray A list of scanners that can be used with ProtoArray Microarrays can be found on the next page The scanner specifications are listed below To acquire ProtoArray data from the image the appropriate microarray data acquisition software is needed The recommended microarray data acquisition software for analysis is GenePix Pro v6 or later Molecular Devices Corporation or ScanAr
139. prepare reagents and buffers e You may use non ionic detergents and reducing agents during probing to minimize non specific interactions e If the protein interaction requires certain co factors be sure to include the co factors in the probing buffer during probing e Prepare the Blocking Buffer and Washing Buffer fresh prior to use e Use the recipes described below to prepare your own buffers Recommended buffers are listed below for blocking and washing the arrays You can perform array probing using the recommended buffers and then based on your initial results optimize the buffer formulation Blocking Buffer final concentration 50 mM HEPES pH 7 5 200 mM NaCl 0 08 Triton X 100 25 Glycerol 20 mM Reduced glutathione 1 mM DTT 1X Synthetic Block 1 Prepare 5 mL of buffer for each microarray For 100 mL Blocking Buffer prepare fresh reagents as follows 1M HEPES pH 7 5 5mL 5M NaCI 4mL 1096 Triton X 100 800 uL 50 Glycerol 50 mL Glutathione powder 610 mg 10X Synthetic Block 10 mL 2 Adjust pH to 7 5 with NaOH 3 Add 100 pL of 1 M DTT 4 Add water to 100 mL Mix well do not vortex and store on ice until use Blocking Buffer without 10X Synthetic Block and DTT may be prepared the day before the assay Store stock at 4 C for no more than 24 hours Continued on next page 21 Protein Protein Interaction Probing Procedure Continued Preparing Washing Buffer Preparing Protein Probes 22 Washing
140. printed on the microarray contain a GST Glutathione S Transferase fusion tag and some proteins also contain polyhistidine 6x tag do not use an anti GST antibody or anti polyhistidine antibody for detecting interactions on a ProtoArray Human Protein Microarray We strongly recommend that you probe the ProtoArray Human Protein Microarray with only your detection reagent to detect signals resulting due to interactions between the detection reagent and proteins printed on the array Due to the large variety of protein probes and detection systems that can be used for probing the ProtoArray Human Protein Microarray it is not possible to have a single probing protocol that is suitable for all proteins and detection systems Use the probing procedure from this section as a starting protocol and based on your initial results empirically determine the probing protocol by optimizing the probe concentration buffer formulation incubation time or detection reagents Optimization of probing protocol can be easily and rapidly achieved using multiple ProtoArray Human Protein Microarrays Before starting the probing procedure make sure you have all items on hand especially buffers pages 21 22 probes in Washing Buffer page 22 LifterSlip coverslips see page 20 and incubation tray see page 20 Make sure the buffers are cold Store buffers on ice until use Place an incubation tray on ice to chill until use Review Important Guidelin
141. r screen for 48 3 hours 6 Scan the developed X ray film or phosphor screen and save 49 an image of the array 7 Download the protein array lot specific information the 49 GAL file from ProtoArray Central Portal to acquire and analyze the data using ProtoArray Prospector to identify protein kinase substrates Continued on next page 35 Experimental Overview Continued Control The experimental workflow for probing a ProtoArray Control Protein Microarray Microarray for KSI with the Control Kinase is shown below Experimental Workflow Control Kinase Control Protein Array Observe Alignment Control Kinase and Control Kinase Substrate Signals Probe Human See _ Protein Troubleshooting Array Continued on next page 36 Experimental Overview Continued Human Microarray The experimental workflow for probing ProtoArray Human Protein Microarray Experimental for KSI with your protein kinase of interest is shown below Workflow Protein Protein Microarray Microarray Kinase Buffer only no kinase Radiolabeled ATP Observe Signals at Alignment Control Kinase Locations Observe Signals at Alignment Control Kinase User Supplied Kinase 50 nM Radiolabeled ATP Locations Observe Substrate Phosphorylation lt gt Identify Phoshorylated Substrates
142. r such that the proteins on the array are facing the laser source Decrease stringency Decrease the number of washes Perform probing and washing in the absence or lower concentration of detergent or salts High background Improper blocking Prepare the Blocking Buffer fresh as described on page 21 Improper washing To obtain the best results perform the recommended washing steps Prepare the Washing Buffer fresh as described on page 22 Array dried during probing Do not allow the array to dry during probing Array not dried properly Dry the array as described on page 27 before before scanning scanning High probe concentration Decrease the probe concentration or decrease the incubation time Antibody cross reactivity Probe a protein array using only the antibody without the protein probe to detect cross reactivity with the antibody only Continued on next page 33 Troubleshooting Continued Uneven background Uneven blocking or During the blocking or washing steps ensure the washing array is completely immersed in blocking solution or Washing Buffer and use at least 5 mL buffer in the Incubation tray to cover the array completely with buffer Improper washing To obtain the best results perform the recommended washing steps Prepare the Washing Buffer fresh as described on page 22 Portions of array have dried Do not allow the array to dry during probing Improper array handling Always wear gloves and avoid t
143. ray For more details on array specifications see pages 4 and 7 For research use only Not intended for human or animal diagnostic or therapeutic uses Overview Introduction Types of Microarrays Introduction The ProtoArray Human Protein Microarray allows rapid and efficient detection of protein interactions using a suitable protein or small molecule probe The ProtoArray Control Protein Microarray provides a rapid sensitive and efficient method to verify probing and detection protocols within a day The ProtoArray technology is based on the yeast protein microarray technology developed by Zhu et al 2001 to detect molecular interactions with proteins The ProtoArray Human Protein Microarray contains thousands of purified proteins printed in duplicate on a nitrocellulose coated glass slide See below for details Two types of ProtoArray Microarrays are currently available e ProtoArray Human Protein Microarray v5 0 see page 4 for details Contains gt 9 000 human proteins expressed using a baculovirus expression system purified from insect cells and printed in duplicate on a nitrocellulose coated glass slide e ProtoArray Control Protein Microarray v5 0 see page 7 for details Contains various controls printed in duplicate on a nitrocellulose coated glass slide Each human and control microarray is available for the following specific applications and includes application specific controls printed on
144. ray Human Protein Microarray e AlphaArray Reader Alpha Innotech Corporation e arrayWoRx 4 Color Biochip Reader Applied Precision LLC e SpotLight TeleChem International Inc The following scanners are not compatible with ProtoArray Human Microarray e GeneChip Scanner 3000 Affymetrix Inc e DNA Microarray Scanner Agilent Technologies Inc Additional scanner recommendations can be found under the Resources link under BioMarker Discovery Resources at www invitrogen com protoarray Continued on next page Scanning Arrays Using a Fluorescence Scanner Continued Scanning Procedure A brief procedure for scanning the ProtoArray Microarrays with a fluorescence microarray scanner is described below For details on using a specific scanner or non fluorescent scanner refer to the manufacturer s manual supplied with the scanner The scanning time for each array is 7 8 minutes 1 Start the appropriate array acquisition and analysis software on the computer connected to the fluorescence microarray scanner Open the microarray enclosure on the scanner Place the ProtoArray Microarray in the holder such that the nitrocellulose coated side of the array faces the laser source and barcode on the array is closest to the outside of the instrument Close the microarray enclosure on the scanner 5 Set the following settings to image the microarray Wavelength Choose the appropriate wavelength base
145. ray Acquisition Software PerkinElmer Inc Insert array into the fluorescence microarray scanner Adjust scanner settings Preview the microarray and adjust settings if needed Scan the microarray Save image data Gne sooo EX Es Export and analyze results The fluorescence microarray scanner specifications required to image the ProtoArray Microarray are listed below Array Compatibility Size Standard 1 x 3 or 25 mm x 75 mm microscope slides Thickness 1mm Detection Light and Detector Facing array Orientation Scanned Area 22 mm x 73 mm Focus Auto focus or adjustable 200 um Excitation Depends on the fluorophore used for detection Detection limit 0 1 fluor uM Resolution lt 10 uM Dynamic Range 23 orders of magnitude Output 16 bit TIFF Continued on next page 123 Scanning Arrays Using a Fluorescence Scanner Continued Recommended Scanners 124 The following scanners are compatible for scanning ProtoArray Human Protein Microarray e GenePix 4000A Molecular Devices Corporation e GenePix 4000B Molecular Devices Corporation e GenePix Professional 4200A Molecular Devices Corporation e GenePix Personal 4100A Molecular Devices Corporation e ScanArray Lite PerkinElmer Inc e ScanArray Express PerkinElmer Inc e ScanArray Express HT PerkinElmer Inc e LS Series Laser Scanner Tecan Group AG The following scanners may be compatible with ProtoAr
146. ray and adjust settings if needed Scan the microarray Save image data 9g Qr me QS Export and analyze results For detailed instructions on Data Acquisition and Analysis refer to page 126 1 To acquire data from the scanned image use the barcode on the array to download the GAL file from ProtoArray Central as described on page 126 2 Use the GAL file and suitable microarray data acquisition software to acquire pixel intensity values for all features on the array 3 Analyze data with ProtoArray Prospector using the guidelines on page 128 to determine significant signals with the controls and your protein probe Continued on next page Scanning and Data Analysis Continued Analyzing ProtoArray Prospector Results The Next Step After data analysis ProtoArray Prospector presents a summary of the analyzed data in a table format see ProtoArray Prospector manual for details The antibodies that score as positive in the experiment are proteins that satisfy the basic program options Review the information on page 120 Expected Results to help with data interpretation We recommend validating the interactions as described below After identifying a positive interaction on the ProtoArray Human Protein Microarray you may validate the protein interaction using the ProtoArray Technology or other methods Using the ProtoArray Technology validate the antibody protein interactions by performing experi
147. re and manual are available for FREE to ProtoArray Microarray users and are accessible online at the ProtoArray Central Portal To download the ProtoArray Prospector software and manual go to www invitrogen com protoarray and click on the Online Tools link under BioMarker Discovery Resources ProtoArray Human Protein Microarray Introduction Human Microarray Specifications Array Specifications The ProtoArray Human Protein Microarray is a high density protein microarray containing thousands of purified human proteins for protein interaction screening Each human open reading frame ORF is expressed as an N terminal GST fusion protein using a baculovirus expression system purified from insect cells and printed in duplicate on a nitrocellulose coated glass slide The human proteins spotted on the microarray are expressed in insect cells using an optimized process to maximize the production of soluble recombinant proteins in a high throughput format Schweitzer et al 2003 Proteins are expressed at high levels in insect cells which are similar to mammalian cells with respect to protein folding and post translational modifications such as phosphorylation and glycosylation Bouvier et al 1998 Hollister et al 2002 Predki 2003 in contrast to proteins expressed in E coli This allows protein interaction detection at a functional level Details on the human microarray are described in this section The ProtoArray
148. rent probe concentration as described below Probe first array And Then Probe Second Array With 10 nM probe Weak signal With 1 10 uM probe With 10 uM probe High background With 10 100 nM probe Continued on next page 15 Guidelines for Probing the ProtoArray Microarray Continued Control The experimental workflow for probing ProtoArray Control Protein Microarray Microarray with Array Control Protein using a fluorescent detection system is shown below Experimental Workflow Array Control Protein Control Array Observe Alexa Fluor Ab and Control Protein Signals See Probe Human Troubleshooting Protein Array Continued on next page 16 Guidelines for Probing the ProtoArray Microarray Continued Human Microarray The experimental workflow for probing the ProtoArray Human Protein Experimental Microarray is shown below Workflow Protein Microarray Block array with Blocking Buffer Probe array with protein probe in Washing Buffer 4 C 90 minutes Biotin and V5 tags are recommended Wash array with Washing Buffer Fluorophore labels such as Alexa Fluor 647 dyes are recommended Protein probe contains a tag Protein probe is labeled No gt Fluororescent detection systems are recommended Perform secondary detection Wash array with Washing Buffer Dry array Scan
149. rified human proteins are printed on nitrocellulose coated slides in a Human dust free and temperature and humidity controlled environment to maintain Proto Array consistent quality of the microarrays The arrays are printed using an automated process on an arrayer that is extensively calibrated and tested for printing ProtoArray Human Protein Microarrays Maintaining ProtoArray Human Protein Microarrays are produced using rigorous Stringent Quality production and quality control procedures with an integrated data management Control system to ensure consistent results and maximize inter and intra lot reproducibility Pre Printing Ouality Control Prior to production the arrayer and supporting components are tested and adjusted to production specifications The quality and performance of pins is critical and all pins are extensively tested and calibrated To maintain protein stability and function arrays are printed at 6 C under controlled environmental conditions Post Printing Quality Control After production each microarray is visually inspected for obvious defects that could interfere with the experimental results The presence of each control and human protein spot is assessed by fluorescent scan of a representative number of arrays and acquisition of signals due to fluorescence of the printing buffer Signal to background ratios SBR are determined for each spot and spots with a SBR less than 3 are labeled missing The
150. roarray The Control Kinase phosphorylates the Control Kinase substrate producing a signal These signals indicate proper probing and scanning procedures Note 52 To orient the results obtained from the GAL file and ProtoArray Prospector with the array image position the microarray image such that the barcode is at the bottom of the image In this orientation the top left corner of the microarray image is Block 1 Continued on next page Expected Results for KSI Continued Human ProtoArray Probing Results The results obtained after probing the ProtoArray Human Protein Microarray vo 0 with 50 nM Control Kinase is shown below The Control Kinase phosphorylates the Control Kinase substrate printed on the array A negative control image of the ProtoArray Human Protein Microarray v5 0 is also shown below Signal spots without captions represent features exhibiting autophosphorylation Image of the Human Microarray when probed with labeled ATP only negative control Image of the Human Microarray when probed with 50 nM Control Kinase Human Array Boxed area shown in Human Array Image Boxed area shown in Alignment Control E Kinase PKCeta Image detail detail Alignment Control j dd Al Kinase EKCeta Control King PKCeta Control Kinase Substrate MAPKAP Alignment Control Kinase PKCeta 53 Troubleshooting
151. roarray with only your detection reagent to detect signals resulting due to interactions between the detection reagent and proteins printed on the array Due to the large variety of protein probes and detection systems that can be used for probing the ProtoArray Human Protein Microarray it is not possible to have a single probing protocol that is suitable for all proteins and detection systems Use the probing procedure from this section as a starting protocol and based on your initial results empirically determine the probing protocol by optimizing the probe concentration buffer formulation incubation time or detection reagents Optimization of probing protocol can be easily and rapidly achieved using multiple ProtoArray Human Protein Microarrays When performing fluorescence detection it is important to avoid exposing the array to light after probing with a fluorescent detection reagent If performing direct labeling always verify that labeling does not affect the binding affinity of the antibody Although Alexa Fluor 555 or Cy3 dyes can be used for detection using them may result in higher background signals Continued on next page Antibody Specificity Profiling Application Probing Procedure Continued Blocking Step Instructions for blocking the microarray are described below 1 Immediately place the mailer containing the ProtoArray Human Protein Microarray v5 0 at 4 C upon removal from storage at 20 C an
152. robe scan the microarray using a suitable microarray scanner After scanning and saving an image of the array download the protein array lot specific information from the ProtoArray Central Portal Use the lot specific information to acquire and analyze the data to identify specific ubiquitination targets Imaging hardware A suitable scanner is required to scan the ProtoArray Microarray The scanner specifications are listed page 123 For a list of scanners to use with ProtoArray Microarrays see page 124 Data acquisition software We recommended GenePix Pro v6 or later Molecular Devices Corporation or ScanArray Acquisition Software PerkinElmer Inc as microarray data acquisition software for analysis of images For detailed instructions on scanning the microarray refer to Scanning Arrays Using a Fluorescence Scanner page 123 Insert array into the fluorescence microarray scanner Adjust scanner settings Preview the microarray and adjust settings if needed Scan the microarray Save image data Drs RS SGI ND ops Export and analyze results For detailed instructions on Data Acquisition and Analysis refer to page 126 1 To acquire data from the scanned image use the barcode on the array to download the GAL file from ProtoArray Central as described on page 126 2 Use the GAL file and suitable microarray data acquisition software to acquire pixel intensity values for all features on the array 3 A
153. rray you need 120 uL of your small molecule probe with a suitable tag The recommended small molecule probe concentration for probing the ProtoArray Human Protein Microarray is at least 2 5 uM If the small molecule is dissolved in an organic solvent such as ethanol or DMSO the final organic solvent concentration should be less than 1 DMSO by volume or 5 ethanol by volume Dilute the probe to the recommended starting concentration in SMI Assay Buffer Mix well do not vortex and store protected from light on ice until use Continued on next page Small Molecule Interaction Probing Procedure Continued Preparing Streptavidin Solution Before Starting Important The biotinylated probe is detected using an Alexa Fluor 647 fluorescent conjugated streptavidin Prepare 5 mL of streptavidin solution for each array to be probed e Biotin detection Prepare 1 ug mL Streptavidin Alexa Fluor 647 Conjugate in SMI Assay Buffer e Before starting the probing procedure make sure you have all items on hand especially buffers previous page probes in SMI Assay Buffer see page 60 LifterSlip coverslips see page 59 and incubation tray see page 59 e Make sure the buffers are cold Store buffers on ice until use Place an incubation tray on ice to chill until use e Review Important Guidelines on page 11 prior to starting the probing procedure e We strongly recommend that you probe the ProtoArray Human Protein Microarray w
154. rray should not produce any positive results using the solution assay Expected Results for KSI Introduction The controls printed on the ProtoArray Microarray are useful in verifying the probing and scanning protocols as described below Control Alignment Control Kinases Control Kinase Description Function Verification Alignment Control The Alignment Control Kinases Proper probing Kinases are printed PKCeta autophosphorylate during and scanning on the microarray the labeling reaction The signals procedures are used as reference spots to orient the microarray image and help assign spot identities The Control Kinase The Control Kinase MAPK14 p38 Proper probing Substrate substrate is printed alpha included in the complete kit and scanning on the microarray phosphorylates the Control Kinase procedures MAPKAD substrate producing a signal GST Protein A GST protein Detects non specific binding to GST Negative Control Gradient gradient is printed on and serves as a negative control the array The signals are also used for background calculation by ProtoArray Prospector software ProtoArray Human Protein Microarrays are designed for kinase substrate Note identification After performing the KSI assay and identifying potential kinase substrates we recommend that you validate the observed substrate phosphorylation using another method such as in vitro solution assay Using ProtoArray Huma
155. see aa De Pe Pe PELA 35 Working with Radioactive Material sse tnter 38 Guidelines for Probing the ProtoArray Microarray eese tenente tentent tete ans 39 Preparing the Protein Kinase sese tenente nnne 40 Kinase Substrate Identification Probing Procedure sse 41 Image Acquisition and Processing sss esr poeti DEEE EA e eren E e E 49 Expected Results for KS jist e oie eoo nhi oe eaea A haere a e Kae E ie eed 51 Troubl shooting sno datant OR tutta RED ed OR tt 54 Small Molecule Identification SMI Fluorescent Application eere 56 Experimental Ovetview u e ert ete Hee te i eee HR te ee HE ah ire see LE ede pra ation 56 Guidelines for Probing the ProtoArray Microattray i oen teer tinet aiino etiatn 57 Preparing the Small Molecule Probe ccccccceccesesesesssneeseseseececeeeceesesesesesnsnsneneseseseseececeneseseseeeeeeeenananes 58 Small Molecule Interaction Probing Procedure essere 59 Scanning and Data Analy Sissin esns ai is AE EEE E ETA AE i ET NE tnnt 66 Scanning and Data Analysis Continued sse 67 Expected Results for SMI Fluorescent tenete 68 Troubleshooting eane eere e eaae eE eE ee AE ubere Ont dae Ute e dietas 69 Tritium Radiolabeled Small Molecule Identification SMI Radioactive Application 71 Experimental Overview ae sente eas sen eta sae ae RE ne en Et et HH e FH e este
156. sensitive phosphor screen Note The tritium sensitive phosphor screen will eventually be damaged due to tritium contamination Directly washing the screen with methanol can remove some contamination However for critical experiments we recommend the use of a new screen or a screen which has been verified to be free of contaminants by pre exposure in an empty cassette for several days followed by scanning and imaging Expose ProtoArray Protein Microarrays to the phosphor screen for 16 days Note For best results we recommend scanning the screen after a minimum of 16 days of exposure However tritium signals have been observed within 24 hours of exposure for some radioligands Proceed to Image Acquisition and Processing next page Image Acquisition and Processing Introduction Materials Needed Scanning the Array Data Acquisition and Analysis Once you have exposed the ProtoArray to the phosphor screen scan the phosphor screen to acquire a TIFF image that is required for microarray data analysis Imaging hardware A phosphorimager that provides at least 50 uM resolution to acquire the image from a phosphor screen see page 129 for phosphorimagers that have been tested with ProtoArray Microarrays Data acquisition software We recommended GenePix Pro v6 or later Molecular Devices Corporation or ScanArray Acquisition Software PerkinElmer Inc as microarray data acquisition software for analysis of images
157. settings if needed Scan the microarray Save image data 9g Qr me QS Export and analyze results For detailed instructions on Data Acquisition and Analysis refer to page 126 1 To acquire data from the scanned image use the barcode on the array to download the GAL file from ProtoArray Central as described on page 126 2 Use the GAL file and suitable microarray data acquisition software to acquire pixel intensity values for all features on the array 3 Analyze data with ProtoArray Prospector using the guidelines on page 128 to determine significant signals with the controls and your protein probe Continued on next page Scanning and Data Analysis Continued Analyzing ProtoArray Prospector Results The Next Step Detecting Reciprocal Interactions After data analysis ProtoArray Prospector presents a summary of the analyzed data in a table format see ProtoArray Prospector manual for details The proteins that score as positive in the experiment are proteins that satisfy the basic program options Review the information on page 30 Expected Results to help with data interpretation We recommend validating the interactions as described below After identifying a positive interaction on the ProtoArray Human Protein Microarray you may validate the protein interaction using the ProtoArray Technology or other methods Using the ProtoArray Technology validate the protein protein interactions by
158. software program if desired 3 After the grid is properly adjusted and all of the features are aligned acquire the pixel intensity data for each feature by clicking the Analyze button in GenePix Pro and save export the results as a GPR GenePix Results file for data analysis using ProtoArray Prospector next page Note If you wish to perform data analysis using Microsoft Excel save export the results with an xls extension or rename the tab or gpr file using the xls extension The ProtoArray Prospector software quickly analyzes the data acquired from the image acquisition software and easily identifies statistically significant interactors saving you time and effort In addition the software has features that allow you to modify the analysis method and compare data obtained from different arrays The ProtoArray Prospector software and manual are available free of charge to ProtoArray Microarray users To download the ProtoArray Prospector software and manual go to www invitrogen com protoarray and click Online Tools link under BioMarker Discovery Resources The ProtoArray Prospector software currently accepts the output files GPR generated by the GenePix Pro microarray data acquisition software and analyzes the data using specified algorithms to generate a list of human proteins showing significant interactions with the probe If GPR files are not available consult the ProtoArray Prospector User Manual for guid
159. t return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing invitrogen com This product is the subject of WIPO patent WO8809372 and foreign equivalents to be used for scientific investigation and research and for no other purpose whatsoever Licenses for commercial use of the above mentioned patents must be negotiated directly with Amrad Corporation 576 Swan Street Richmond Victoria Australia 3121 Phone 61 3 9208 4000 Continued on next page Purchaser Notification Continued Limited Use Label License No 221 Microarrays of Biological Samples Limited Use Label License No 240 Protein Array Content Limited Use Label License No 295 Polypeptides Expressed in Yeast This product may be covered by one or more of U S Patent numbers 5 807 522 and 6 110 426 licensed exclusively to Incyte Corporation The purchase of this product conveys to the buyer whether employed in academia government not for profit entity or a for profit entity the limited non exclusive non transferable right without the right to resell repackage or further sublicense under these patents to use this product for research and development purposes No other license is granted to the buyer whether expressly
160. tails The proteins that score as positive in the experiment are proteins that satisfy the basic program options We recommend that candidate biomarkers be validated in a follow on experiment using ProtoArray or other methods There are several appropriate assay formats including ELISA Luminex and immunoblotting Continued on next page 105 Expected Results for IRBP Introduction The controls printed on the ProtoArray Human Protein Microarray are useful in verifying the probing detection and scanning protocols as described below Control The results obtained after probing the ProtoArray Control Protein Microarray ProtoArray v5 0 v5 0 for IRBP with 1 500 diluted human serum and Alexa Fluor 647 goat anti Probing Results human IgG antibody are shown below Human Array Image Boxed Area shown in detail ee Cee Alexa eel Alexa Human IgG Fluor Ab Fluor Ab se Anti human IgG Alexa Fluor Ab V5 Control Protein N oe The following control features can be observed after probing a ProtoArray Protein Microarray e Alexa Fluor Ab signal This is an antibody labeled with Alexa Fluor 647 The fluorescent antibody signals indicate that the array has been properly scanned and are used as reference spots to orient the microarray and help assign spot identities e Human IgG Signal A protein gradient of purified human IgG is printed on each subarray and serves as a positive control whe
161. tain co factors be sure to include the co factors in the kinase buffer during probing Continued on next page Kinase Substrate Identification Probing Procedure Continued Preparing 0 5 SDS Preparing KSI Blocking Buffer e To perform the washing and probing steps we recommend using a sterile 50 mL conical tube e Incubation trays or other hybridization chambers may not be suitable for use as you need a container that seals tightly to prevent any leakage of radioactive material during the washing steps e Do not use any cold ATP for the kinase probing steps If your kinase is stored in a buffer containing ATP make sure the final concentration of cold ATP is less than 100 nM during the kinase probing step e Avoid adding more than 10 v v of your protein kinase sample to 120 pL of Kinase Buffer Addition of more than 10 of your kinase to the Kinase Buffer can decrease the assay performance Prepare 80 mL of 0 5 SDS for each microarray For 200 mL 0 5 SDS prepare the following reagents fresh from 10 SDS as follows 10 SDS 10 mL Ultrapure water 190 mL Total Volume 200 mL Mix well and store at room temperature until use KSI Blocking Buffer final concentration 1X PBS 1 BSA 1 Prepare 5 mL of buffer for each microarray For 100 mL KSI Blocking Buffer prepare fresh reagents as follows 10X PBS pH 7 4 10 mL 30 protease free BSA 3 3 mL Deionized water to 100 mL 2 Mix well do not vortex 3 Sterile f
162. tal outline for performing SMI application using the ProtoArray Steps Human Protein Microarray with tritium radiolabeled small molecules is shown below Step Action Page no 1 Block ProtoArray Human Protein Microarray with 5 mL 76 Tritium SMI Assay Buffer with gentle agitation at 4 C 2 Probe ProtoArray Human Protein Microarray with 100 pL 77 of H labeled small molecule in Tritium SMI Assay Buffer Optional If you are a first time user of the ProtoArray Human Protein Microarray perform a control probing using a ProtoArray Control Protein Microarray to verify the assay protocol Dry the microarray 78 Expose the microarray to tritium sensitive phosphor screen 78 phosphor screen for 16 days Scan phosphor screen with phosphorimager 79 Download the protein array lot specific information the 79 GAL file from ProtoArray Central Portal to acquire and analyze the data using ProtoArray Prospector to identify small molecule substrates Continued on next page 71 Guidelines for Probing the ProtoArray Microarray Introduction Control Protein Microarray Probing Options Human Protein Microarray Probing Options 72 The ProtoArray tritium labeled small molecule profiling application has adequate sensitivity to identify target protein interactions with a Kd of 10uM The minimum specific activity of the small molecule should be at least 10 Ci mmol and weaker interactions may r
163. temperature e Review Important Guidelines on page 11 and Working with Radioactive Material on page 38 prior to starting the probing procedure 45 Kinase Substrate Identification Probing Procedure Continued Blocking Step 46 Instructions for blocking the ProtoArray Microarray are described below 1 Remove the mailer containing the ProtoArray Microarray from storage at 20 C and place immediately at 4 C be sure to use the microarray before the expiration date printed on the box Allow the array to equilibrate in the mailer at 4 C for at least 15 minutes prior to blocking Failure to do so may result in condensation on the array Place one microarray with the barcode facing up into each well of a chilled 4 chamber incubation tray such that the barcoded end of the microarray is near the end of the tray marked with an indented numeral see figures 1a and 1b below Using a sterile pipette immediately add 5 mL KSI Blocking Buffer into each chamber containing an array Avoid pipetting buffer directly onto the array surface Incubate the tray for 1 hour at 4 C on a shaker set at 50 rpm circular shaking After incubation remove array from 4 chamber incubation tray using forceps Insert the tip of the forceps into the indentation at the numbered end of the tray and gently pry the array upward see figure 2 below Using a gloved hand pick up the microarray by holding the array by its edges only Tap to remove
164. the array e Protein Protein Interaction PPI e Kinase Substrate Identification KSI e Small Molecule Protein Interaction SMI Profiling Fluorescent and Radioactive e Immune Response Biomarker Profiling IRBP e Ubiquitin Ligase Profiling e Antibody Specificity Profiling ASP application Continued on next page Overview Continued Applications Advantages The ProtoArray Microarray allows you to Detect novel protein protein interactions Validate previously observed protein protein interactions for PPI applications Jin et al 2006 Satoh et al 2006 or observed signals for KSI applications Mah et al 2005 Ptacek et al 2005 Boyle et al 2007 Confirm positive interactions using the identified interacting protein on the array as a probe in reciprocal experiments page 29 Test various experimental conditions for the protein interactions or your kinase Rapidly perform serum profiling using a sensitive method to detect potential autoantigen biomarkers Mattoon et al 2005 Michaud et al 2003 Identify potentially biologically relevant protein kinase substrates small molecule binding partners ubiquitin ligase substrates and protein interactors of research or therapeutic antibodies Using the ProtoArray Human Protein Microarrays to detect protein interactions offers the following advantages Provides a simple rapid sensitive and efficient method to identify protein interactions within a day Allo
165. the protein to obtain the best results Epitope tags such as FLAG myc or HA can also be used for probing the microarray in conjunction with an appropriate labeled antibody Note Do not use an anti GST antibody or anti polyhistidine antibody for detecting interactions on a ProtoArray Protein Microarray as the majority of proteins on the array are GST tagged with some that are also polyhistidine tagged The extremely high affinity of the biotin streptavidin interaction makes biotin protein conjugation an attractive method for probe labeling Small amounts of the protein can be efficiently in vitro biotinylated in a simple procedure The biotinylated protein probe is detected using a streptavidin detection system Epitope Tag To generate your protein probe with an epitope tag you need to express your protein of interest as a fusion protein in an expression vector containing the desired epitope tag at the N or C terminus of the protein A variety of vectors with different tags are available from Invitrogen for expression of your protein of interest For more information about these products refer to our website www invitrogen com or call Technical Support page 137 The recommended epitope tag for use with the ProtoArray Human Protein Microarray is the V5 epitope tag Biotin Tag You may use any method to in vitro biotinylate your protein of interest We recommend using the Biotin XX Microscale Protein Labeling Kit from Invitroge
166. the results as a GPR GenePix Results file for data analysis using ProtoArray Prospector see next page The results contain the pixel intensity information for each spot feature on the array and information on additional parameters depending on the type of software used for data acquisition Note Do not use the automatic feature finding function in GenePix while acquiring data from a radiometric assay For other microarray data acquisition software use data from the GAL files from ProtoArray Central to generate files that are compatible with your microarray data acquisition software to define the microarray grid Note If you wish to perform data analysis using Microsoft Excel save export the results with an xls extension or rename the tab or gpr file using the xls extension Continued on next page 133 Data Acquisition and Analysis Continued Data Analysis Using ProtoArray Prospector 134 The ProtoArray Prospector Analyzer software quickly analyzes the data acquired from the ProtoArray Prospector Imager or image acquisition software and easily identifies statistically significant hits saving you time and effort The Analyzer software is designed to analyze data and identify potential protein binding partners with a low false positive rate as compared to performing manual calculations using a spreadsheet program In addition the software has features that allow you to modify the analysis method and compar
167. tion tray on ice to chill until use Review Important Guidelines on page 11 prior to starting the probing procedure We strongly recommend that you probe the ProtoArray Human Protein Microarray with only Biotin Ubiquitin and your detection reagent to detect signals resulting due to interactions between the detection reagent and proteins printed on the array You may also want to probe an array in the absence of the E3 ligase Due to the large variety of protein probes and detection systems that can be used for probing the ProtoArray Human Protein Microarray it is not possible to have a single probing protocol that is suitable for all proteins and detection systems Use the probing procedure from this section as a starting protocol and based on your initial results empirically determine the probing protocol by optimizing the probe concentration buffer formulation incubation time or detection reagents Optimization of probing protocol can be easily and rapidly achieved using multiple ProtoArray Human Protein Microarrays Instructions for blocking the microarray are described below Immediately place the mailer containing the ProtoArray Human Protein Microarray v5 0 at 4 C upon removal from storage at 20 C and equilibrate the mailer at 4 C for at least 15 minutes prior to use Place ProtoArray Human Protein Microarrays with the barcode facing up in the bottom of a 4 chamber incubation tray such that the barcode en
168. tored either vertically or horizontally After drying store the arrays vertically or horizontally in a slide box protected from light Avoid prolonged exposure to light as it will diminish signal intensities To obtain the best results scan the array within 24 hours of probing Proceed to Scanning Arrays next page 117 Scanning and Data Analysis Introduction Materials Needed Scanning the Array Data Acquisition and Analysis 118 Once you have probed the ProtoArray with your antibody scan the microarray using a suitable microarray scanner After scanning and saving an image of the array download the protein array lot specific information from the ProtoArray Central Portal Use the lot specific information to acquire and analyze the data to identify specific antigen targets Imaging hardware A suitable scanner is required to scan the ProtoArray Microarray The scanner specifications are listed page 123 For a list of scanners to use with ProtoArray Microarrays see page 124 Data acquisition software We recommended GenePix Pro v6 or later Molecular Devices Corporation or ScanArray Acquisition Software PerkinElmer Inc as microarray data acquisition software for analysis of images For detailed instructions on scanning the microarray refer to Scanning Arrays Using a Fluorescence Scanner page 123 Insert array into the fluorescence microarray scanner Adjust scanner settings Preview the microar
169. ts for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Invitrogen Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accep
170. ty and function arrays are printed at 6 C under controlled environmental conditions Post Printing Quality Control After production each microarray is visually inspected for obvious defects that could interfere with the experimental results The arrays are also functionally qualified by probing control proteins to detect the appropriate interactions Continued on next page Control Proteins ProtoArray Control Protein Microarray Continued Various proteins and controls are printed on each ProtoArray Human Protein and Control Protein Microarray to allow you to verify reagents background and detection conditions used during probing The table below lists the controls printed on each ProtoArray Microarray Protein Function Control Spots required for PPI SMI Fluorescent IRBP ASP and Ubiquitin Ligase Data Analysis Alexa Fluor Antibody Serves as a positive control for fluorescence scanning and for Rabbit anti mouse IgG Antibody labeled with Alexa Fluor 647 Alexa Fluor 555 and Alexa Fluor 488 orientation of the microarray image Bovine Serum Albumin BSA A negative control for non specific protein interactions Biotinylated Anti mouse Antibody A positive control for interaction with streptavidin labeled detection reagent Anti biotin Antibody mouse anti biotin antibody Detects biotin labeled protein probes and serves as a control for anti mouse antibody detection reagent
171. ube to dislodge the coverslip Do not remove the coverslip with forceps if the coverslip does not float away from the array Using forceps carefully remove the dislodged coverslip without touching the array surface Discard the coverslip appropriately as radioactive waste Cap the conical tubes and incubate arrays in 0 5 SDS for 15 minutes at room temperature Note Perform all washing steps with SDS and water without shaking to prevent any spillage of radioactive waste Decant the 0 5 SDS Discard the wash properly as radioactive waste Slowly add 40 mL 0 5 SDS to the tube dispense SDS as described in Step 8 cap the tube and incubate for 15 minutes at room temperature Decant the 0 5 SDS Discard the wash properly as radioactive waste Add 40 mL ultrapure water to the tube dispense water as described in Step 8 cap the tube and incubate the array for 15 minutes at room temperature Decant the water Discard the wash properly as radioactive waste Add 40 mL ultrapure water to the tube cap the tube and incubate the array for 15 minutes at room temperature Decant the water Discard the wash properly as radioactive waste Proceed to Drying and Exposing the Array below Remove the array from the tube at the end of the probing procedure Briefly tap one edge of the array gently on a laboratory wipe to drain excess buffer Place each array in a slide holder or a sterile 50 mL conical tube if you do not have a slide ho
172. uble and active in buffers used for probing the microarray see recipe on page 44 e Use 120 uL of your purified protein kinase at a recommended final protein concentration of 50 nM to probe each ProtoArray Microarray Kinase Substrate Identification Probing Procedure Recommended Workflow Important The recommended workflow for probing the ProtoArray Human Protein Microarray is described below The recommended protein kinase concentration for probing each array is 50 nM 1 Probe two ProtoArray Human Protein Microarrays simultaneously as follows e Probe the first array using your kinase supplied by the user at 50 nM in the presence of radiolabeled y P ATP to identify potential substrates e Probe the second array using only buffer and no kinase negative control in the presence of radiolabeled j P ATP to determine which signals are specific to your kinase If you are using a ProtoArray Control Protein Microarray probe the control microarray as follows e Probe the control array using the Control Kinase MAPK14 p38 alpha available separately from Invitrogen see page 135 at 50 nM in the presence of radiolabeled y P ATP to verify the probing protocol OR e Probe the control array using your kinase of interest 50 nM in the presence of radiolabeled Y P ATP to assess the performance of your kinase with the array surface and control proteins printed on the array After the probing procedure expose arrays to
173. uitable detection system Optional If you are a first time user of the ProtoArray Human Protein Microarray perform a control probing using a ProtoArray Control Microarray to verify probing and detection protocols 26 Dry the microarray 27 Scan the microarray using a suitable microarray scanner and save an image of the array 28 Download the protein array lot specific information GAL file from ProtoArray Central Portal to acquire and analyze the data using ProtoArray Prospector to identify significant protein protein interactions 28 12 Continued on next page Experimental Overview Continued Experimental The experimental workflow for performing the PPI application using the Workflow ProtoArray Microarray with your protein probe labeled with a suitable tag is shown below Purify Protein protein probe with a suitable tag Quantify protein Probe Proteome Array Do have enough protein Observe Alexa Fluor Ab signals Confirm presence of the tag See troubleshooting Observe protein interactions Protein has the tag Probe another protein array with higher probe Confirm interaction by performing reciprocal interaction with another protein array or using another method of choice concentration 13 Guidelines for Probing the ProtoArray Microarray Introduction Detection
174. ular weight of your protein The protein must be gt 15 kDa to avoid loss during removal of free biotin For proteins purified using metal chelating column chromatography ProBond resin or Ni NTA resin perform dialysis against two changes of PBS to significantly lower the imidazole concentration Low concentrations lt 0 1 of sodium azide or thimerosal in the protein solution have no effect on the biotinylation reaction 19 Protein Protein Interaction Probing Procedure Introduction After purifying the protein probe and verifying the presence of the tag or label on the protein probe the ProtoArray Human Protein Microarray using your protein probe Instructions are included in this section to probe the ProtoArray Human Protein Microarray using your own buffers see page 21 22 for buffer recipes Experimental Block the ProtoArray Human Protein Microarray 1 Outline 2 Probe with your tagged protein probe 3 Perform detection using an appropriate detection system 4 Dry the array for scanning Materials Needed e ProtoArray Human or Control Protein Microarray v5 0 page 135 e Blocking Buffer and Washing Buffer page 21 22 for recipes e Protein probe containing a suitable tag in Blocking Buffer page 21 for recipes e Appropriate Alexa Fluor 647 conjugate or equivalent page 135 keep on ice in dark until immediately before use e Antibody against the epitope tag for an epitope tagged protein probe e Ice
175. urosporin Array Image Boxed Area shown in detail Alexa LI Fluor Ab CAMK2A The following control features can be observed after probing a ProtoArray Protein Microarray 68 Alexa Fluor Ab signal This is an antibody labeled with Alexa Fluor 647 The fluorescent antibody signals indicate that the array has been properly scanned and are used as reference spots to orient the microarray and help assign spot identities CAMK2A signal Staurosporin is a known binding partner for calmodulin kinase CAMK2A and binds to the calmodulin kinase printed on the array The signal is used to verify the probing procedure Troubleshooting Introduction Problem SMI Array Results Weak or no signal with protein probe The table below provides some solutions to possible problems you might encounter when using the ProtoArray Microarray for the SMI Fluorescent application Review the expected results section pages 68 to verify the probing detection and scanning procedures are performed correctly Based on the initial results you may need to optimize the probing and detection protocol by optimizing the probe concentration buffer formulation incubation time or detection reagents Epitope tag not present Confirm the presence of the tag by appropriate assay Poor biotinylation of protein Make sure the small molecule is in a buffer that does not contain any primary amines such as ammonium ions Tris g
176. using a slide holder dip the array into a 50 mL conical tube filled with room temperature distilled water three times Centrifuge the array in the slide holder or 50 mL conical tube at 200 x g for 1 minute in a centrifuge equipped with a plate rotor if you are using the slide holder at room temperature Verify the array is completely dry After slides have been probed and dried they can be stored either vertically or horizontally After drying store the arrays vertically or horizontally in a slide box protected from light Avoid prolonged exposure to light as it will diminish signal intensities To obtain the best results scan the array within 24 hours of probing Proceed to Scanning and Data Analysis next page 65 Scanning and Data Analysis Introduction Materials Needed Scanning the Array Data Acquisition and Analysis 66 Once you have probed the ProtoArray with your small molecule scan the microarray using a suitable microarray scanner After scanning and saving an image of the array download the protein array lot specific information from the ProtoArray Central Portal Use the lot specific information to acquire and analyze the data to identify small molecule interactions Imaging hardware A suitable scanner is required to scan the ProtoArray Microarray The scanner specifications are listed page 123 For a list of scanners to use with ProtoArray Microarrays see page 124 Data acquisition software
177. ution Incorrect phosphorimager Be sure the phosphorimager is capable of used providing at least 50 uM resolution Improper handling of arrays Be sure to allow the mailers with arrays to equilibrate at 4 C for at least 15 minutes prior to use Improper covering of arrays Properly cover the array with a single layer of clear plastic wrap without any creases Continued on next page 82 Troubleshooting Continued High background Improper blocking Prepare the Tritium SMI Assay Buffer fresh as described on page 74 Improper washing For the best results perform the recommended washing steps using Tritium SMI Assay Buffer as outlined in the protocol Array dried during Do not allow the array to dry during probing or probing or washing washing procedure Ensure the coverslip completely covers the printed area of the array During the incubation step at 30 C make sure the 50 mL conical tube is capped to minimize drying During all wash steps ensure the array is completely covered in buffers Array not dried properly Dry the array as described before scanning before scanning High small molecule Decrease the small molecule concentration specific concentration activity or decrease the incubation time Uneven background Uneven blocking or During the blocking or washing steps ensure the washing array is completely immersed in buffers and use at least 40 mL buffer in the 50 mL conical tube to cover the array com
178. v Observe Substrate Phosphorylation T Probe another See Troubleshooting protein microarray with higher concentration of your protein kinase Identify Phosphorylated Substrates i Probe another protein microarray with the same higher or lower concentration of your kinase or another kinase Probe another protein microarray with the same higher or lower concentration of your kinase or another kinase 37 Working with Radioactive Material Introduction General Guidelines Important 38 This section provides general guidelines and safety tips for working with radioactive material For more information and specific requirements contact the safety department of your institution Use extreme caution when working with radioactive material Follow all federal and state regulations regarding radiation safety For general guidelines when working with radioactive material see below Follow these general guidelines when working with radioactive material Do not work with radioactive materials until you have been properly trained Wear protective clothing vinyl or latex gloves and eyewear and use a radiation monitor Work in areas with equipment and instruments that are designated for radioactive use Plan ahead to ensure that all the necessary equipment and reagents are available and to minimize exposure to radioactive
179. ws screening of your protein or small molecule of interest against thousands of human proteins representing multiple gene families such as kinases membrane associated proteins cell signaling proteins and metabolic proteins Built in controls printed on each array to control for background and detection Arrays compatible with most commercially available fluorescence microarray scanners for PPI SMI fluorescent ASP ubiquitin ligase profiling and IRBP signals or autoradiography and phosphorimaging for KSI and SMI radioactive signals Continued on next page Overview Continued ProtoArray Central Portal ProtoArray Prospector The ProtoArray Central Portal provides a web based user interface to access ProtoArray specific information including various applications resources and online tools You can also use the portal to retrieve ProtoArray Lot Specific Information page 126 which is required for analyzing the array data and identifying statistically significant interactions To visit the portal go to www invitrogen com protoarray The ProtoArray Prospector software quickly analyzes the microarray data acquired from the image acquisition software and easily identifies significant hits saving you time and effort In addition the software has features that allow you to modify the analysis method and compare data obtained from different microarrays of the same version number The ProtoArray Prospector softwa

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