Manual - RayBiotech, Inc.
Contents
1. Remove any bubbles on array surfaces Incubate arrays with gentle rocking or shaking for 2 hours at RT or overnight at 4 C Note Avoid the flow of sample into neighboring wells 13 14 Based on number of samples and remaining protocol calculate the amount of 1X Wash Buffer and 1X Wash Buffer Il needed to complete the experiment Separately dilute the required amounts of 20X Wash Buffer Concentrate Item G 20 fold and 20X Wash Buffer Il Concentrate Item H with ddH20 Decant the samples from each well and wash 3 times with 800 ul of 1X Wash Buffer at RT with gentle rocking or shaking for 5 min per wash RayBio L Series Human Antibody Array L 182 Protocol 12 15 Obtain a clean container e g pipette tip box or slide staining jar place the Assembled Glass Slide into the container with enough volume of 1X Wash Buffer to completely cover the entire assembly and remove any bubbles in wells Wash 2 times at RT with gentle rocking or shaking for 10 min per wash 16 Decant the Wash Buffer from each well place the Assembled Glass Slide into the container with enough volume of 1X Wash Buffer II to completely cover the entire assembly and remove any bubbles in wells Wash 2 times at RT with gentle rocking or shaking for 5 min per wash 17 Prepare 1X Cy3 Conjugated Streptavidin a Briefly spin down tube containing the Cy3 Conjugated Streptavidin Item I immediately before use b Add 1000 ul of Blocking Buffe2. 3 129 141 Liu T Xue R Dong L et al Rapid determination of serological cytokine biomarkers for hepatitis B virus related hepatocellulare carcinoma using antibody arrays Acta Biochim Biophys Sin 2011 43 1 45 51 Cui J Chen Y Chou W C et al An integrated transcriptomic and computational analysis for biomarker identification in gastric cancer Nucl Acids Res 2011 39 4 1197 1207 Jun Zhong et all Temporal Profiling of the Secretome during Adipogenesis in Humans Journal of Proteome Research 2010 9 5228 5238 Chowdury UR Madden BJ Charlesworth MC Fautsch MP Proteomic Analysis of Human Aqueous Humor Invest Ophthalmol Visual Sci 2010 51 10 4921 4931 Wei Y Cui C Lainscak M et al Type specific dysregulation of matrix metalloproteinases and their tissue inhibitors in end stage heart failure patients relationshp between MMP 10 andLV remodeling J Cell Mol Med 2011 15 4 773 782 Kuranda K Berthon C Lep tre F et al Expression of CD34 in hematopoietic cancer cell lines reflects tightly regulated stem progenitor like state J Cell Biochem 2011 112 5 1277 1285 Toh HC Wang W W Chia WK et al Clinical Benefit of Allogenic Melanoma Cell Lysate Pulsed Autologous Dendritic Cell Vaccine in MAGE Positive Colorectal Cancer Patients Clin Chem Res 2009 15 7726 7736 RayBio L Series Human Antibody Array L 182 Protocol 22 Zhen Hou Cytokine array analysis of peritoneal fluid between women w
3. according to homogenizer manufacturer instructions 3 Transfer extracts to microcentrifuge tubes and centrifuge for 20 min at 13 000 rpm 4 C Note If the supernatant appears to be cloudy transfer the supernatants to a clean tube centrifuge again at 13 000 rpm for 20 minutes at 2 8 C If the supernatant is still not clear store the lysate at 20 C for 20 minutes Remove from the freezer immediately centrifuge at 13 000 rpm for 20 minutes at 2 8 C 4 Transfer supernatant to a clean tube and store at 80 C B Handling the Glass Slides e The microarray slides are delicate Please do not touch the array surface with pipette tips forceps or your fingers Hold the slides by the edges only e Handle the slides with powder free gloves and in a clean environment e Do not remove the glass slide from the chamber assembly until step 20 on page 14 and take great care not to break the glass slide when doing so e Remove reagents sample by gently applying suction with a pipette to corners of each chamber Do not touch the printed area of the array only the sides as seen in image below RayBio L Series Human Antibody Array L 182 Protocol 6 C Layout of Human L 182 Glass Slide Two or four identical sub arrays on one slide 2 Subarray 4 Subarray Blank D Incubations and Washes e Cover incubation chamber with a Plastic Adhesive Strip Item J to prevent evaporation during incubation or wash steps particularly t
4. 2 Protocol A Dialysis of Sample Note Samples must be dialyzed prior to biotin labeling Steps 5 7 1 To prepare dialysis buffer 1X PBS pH 8 0 dissolve 0 6 g KCI 24 g NaCl 0 6 g KH2PO and 3 45 g Na HPO in 2500 ml de ionized or distilled water Adjust pH 8 0 with 1M NaOH and adjust final volume to 3000 ml with ddH 0O 2 Add each sample into a separate Dialysis Tube Item A Loading volumes are as follows 200 ul cell culture supernatant 100 ul cell or tissue lysate 172 mg ml total protein or 20 ul serum or plasma 80 ul 1X PBS pH 8 5 fold dilution Carefully place Dialysis Tubes into Floating Dialysis Rack Note for cell culture supernatants if using a 2 fold dilution of biotin labeled sample in the array incubation step page 12 step 11 you will need to load a total of 400 ul of original cell culture supernatant into 2 separate Dialysis Tubes 200 ul tube Note If the samples appear to be cloudy transfer the samples to a clean tube centrifuge at 13 000 rpm for 20 minutes at 2 8 C If the samples are still not clear store them at 20 C for 20 minutes Remove from the freezer immediately centrifuge at 13 000 rpm for 20 minutes at 2 8 C 3 Place Floating Dialysis Rack into 2500 ml dialysis buffer in a large beaker Place beaker on a stir plate and dialyze for at least 3 hours at 4C Stirring buffer gently Then exchange the 1X PBS buffer and repeat dialysis for at least 3 h at 4 C Transfer dialyzed sa
5. 2 human target proteins can be simultaneously detected including cytokines chemokines adipokines growth factors angiogenic factors proteases soluble receptors soluble adhesion molecules and other proteins in cell culture supernatants serum and plasma The first step in using the RayBio L Series Human Obesity Antibody Array 182 is to biotinylate the primary amine of the proteins in serum or plasma samples cell culture supernatants cell lysates or tissue lysates The glass slide arrays are then blocked just like a Western blot and the biotin labeled sample is added onto the glass slide which is pre printed with capture antibodies and incubated to allow for interaction of target proteins Streptavidin conjugated fluorescent dye Cy3 equivalent is then applied to the array Finally the glass slide is dried and laser fluorescence scanning is used to visualize the signals RayBio L Series Human Antibody Array L 182 Protocol 2 Il Materials Provided A Storage Recommendations Upon receipt the kit should be stored at 20 C until needed Please use within 6 months from the date of shipment After initial use remaining reagents should be stored at 4 C and may be stored for up to 3 months Labeling Reagent Item B should be prepared fresh each time before use Unused glass slides should be kept at 20 C and repeated freeze thaw cycles should be avoided slides may be stored for up to 6 months ITEM DESCRIPTION AAH B
6. HS4 e404 404 01494 01493 s ervna waa waa 393 393 wa ma 9 uneisho duo epnded o pnded o IND INO v OW aruana env vidawa ENV Wedd srana srana sana Bawa zawa Sawa Sawa sana vana vana gody nd tdv PUNY PUNY E1LdONV 1LdDNV ZILA NY TUdONV HudoNv HudoNv 201924 ay j uy 201924 Dilj Buy I uisuajojBuy uaBoulsuajoibuy isuajoibuy uaBoulsuajoibuy z aav HLV Hav zaw Taw E109 39V 709 30V N DAN 880d E 504 5094 80d sod E L st vi Er z u oL 6 g Zz g s v E z L dew 1 Aeary Apoqnuy suryodipy ueumny poseq pPqeI unorg orgAey 16 L Series Human Antibody Array L 182 Protocol RayBio VI Interpretation of Results A Explanation of Controls Spots 1 2 Positive Control spots POS1 POS2 POS3 are standardized amounts of biotinylated IgGs printed directly onto the array All other variables being equal the Positive Control intensities will be the same for each sub array This allows for normalization based upon the relative fluorescence signal responses to a known control much as housekeeping genes or proteins are used to normalize results in PCR or Western blots respectively Negative Control NEG spots contain a protein containing buffer used to dilute antibodies printed on the array Their signal intensities represent non specific binding of Biotin conjugated anti Cytokines and or the Cy3 Conjugated Streptavidin Negative control signal intensities are usual
7. LG ADI 1 2 AAH BLG ADI 1 4 A Dialysis Vials amp Floating Dialysis Rack 4 vials 8 vials B Labeling Reagent 1 vial 2 vials D Stop Solution 1 vial 50 ul RayBio L Series Human Antibody E Arrade Glace slide 1 slide L 182 1 slides L 182 F Blocking Buffer 1 bottle 8 ml 1 bottle 8 ml G 20X Wash Buffer 1 bottle 30 ml 1 bottle 30 ml H 20X Wash Buffer II 1 bottle 30 ml 1 bottle 30 ml Cy3 Conjugated Streptavidin 1 vial 2 vials J Adhesive Plastic Strips K Labeling Buffer 1 bottle 8 ml n a 2X Cell Lysis Buffer 1 bottle 10 ml 1 tube M 30 ml Centrifuge Tube Each slide contains 2 or 4 identical subarrays HiLyte Plus 555 Only needed if testing cell or tissue lysates B Additional Materials Required e KCI NaCl KH PO Na HPO and ddH O0 e 1mltube small plastic or glass containers e Orbital shaker or oscillating rocker e Beaker stir plate and stir bar e Pipettors pipette tips and other common lab consumables e Laser scanner for fluorescence detection list available online e Aluminum foil RayBio L Series Human Antibody Array L 182 Protocol 3 HI Overview and General Considerations A Preparation and Storage of Samples 1 Preparation of Cell Culture Supernatants 1 2 3 Seed cells at a density of 1x10 cells in 100 mm tissue culture dishes Culture cells in complete culture medium for 24 48 hours Replenish with serum free or l
8. RayBio Label Based L Series Human Obesity Antibody Array 182 L 182 Patent Pending Technology User Manual Revised September 9 2014 For the simultaneous detection of the relative expression of 182 L 182 human obesity proteins in serum plasma cell culture supernatants cell tissue lysates or other body fluids L Series Human Antibody Array L 182 Cat AAH BLG ADI 2 2 Sample Kit Cat AAH BLG ADI 4 4 Sample Kit Please read manual carefully before starting experiment RayBiotech Inc Hir the protein array pioneer company Your Provider for Excellent Protein Array Systems and Services Tel Toll Free 1 888 494 8555 or 1 770 729 2992 Fax 1 770 206 2393 Website www raybiotech com Email info raybiotech com TABLE OF CONTENTS l Introduction and How It WOr KS 2 Il Materials Provided 3 A Storage Recommendation S 3 B Additional Materials Required 3 Hl Overview and General Considerations 4 A Preparation and Storage Of Samples 4 B Handling the Glass Slides 6 C Layout of Human L 182 Slide 7 D Incubation ANd WasheS wwwwwwwmwmsmsmmimmsmmmmmim 7 IV HPO OCC byes tesrtotaeite isi ea eden teen mimi 8 A Dialysis Of SAMPI oc cmnnnnnnnnntnnnnnnnnnnniunnnnnnnns 9 B Biotin Labeling Of Sample 10 C Drying of the Glass Slide 11 D Blocking ANd INCUBATIONS connie 12 E Fluorescence Detection 15 V Antibody Array Maps and Target Lists 16 A RayBio Human Ant
9. actice assembling the device with a blank glass slide 1 2 3 Apply slide to incubation chamber barcode facing upward image A Gently snap one edge of a snap on side image B Gently press other of side against lab bench and push in lengthwise direction image C Repeat with the other side image D RayBio L Series Human Antibody Array L 182 Protocol 15 V Antibody Array Map 180d 180d 750d 2504 Esod Esod DIN 9N IN DIN uvaax uvaax 493A EL vuu vuu wa ED epi 40198 anss1L ara 10190 enssiL vanis vani Sanu Em zan zami p uipuodsoqwos zi ueoapuAs ueoopuks ujuo10195 ujuojo9s vevwas vevwas raas raas ws ws orv 0015 oly 0015 6v 8v 0015 90015 mn Had Tad 914N 7 ZBuvdd 914N 7 zBuvdd curenued eurenued 303d 303d T 4904 T4904 9 450d 94904 8v 4904 av4904 vvd or y uixa1o y uxa 6 4d9 4 uneisego 66 449 4 uneisago A apndadosnan AdN 4 pndedosnen AdN Y 49N Y 49N z uniydonay z uiydoanay LaonaN LaonaN dIVN dIVN UNEISOAN 6 Adi gi EL EL ELJ ELI 259 W 259 W dOW EdW L d3W t dOW upejoydwk unoeoydwiq xo1 8 unda undaT 02209 4 unsur 02209 4 ulinsuj ujinsu unsur HYSNI HYSNI 7SNI ISNI IL 71 SZ ILLT 57 01 zA zA LT L vaa vara w w 1491 W491 da491 48491 748491 748491 1748491 148491 us F491 us kadi hadi 9 Lasepmosdauomienio 1 se
10. allow the Assembled Glass Slide to equilibrate to RT 9 Open package and take the Assembled Glass Slide out of the sleeve Do not disassemble the Glass Slide from the chamber assembly Place glass slide assembly in laminar flow hood or similar clean environment for 1 2 hours at RT Note Protect the slide from dust or other contaminants RayBio L Series Human Antibody Array L 182 Protocol 11 D Blocking and Incubations Note Glass slide should be completely dry before adding Blocking Buffer to 10 11 Note 12 wells Block sub arrays by adding 400 ul of Blocking Buffer Item F into each well of Assembled Glass Slide and incubating at RT for 30 min Ensure there are no bubbles on the array surfaces Immediately prior to sample incubation spin biotin labeled samples for 5 min at 10 000 rpm to remove any particulates or precipitates Dilute samples with Blocking Buffer Recommended dilution of the biotin labeled samples with Blocking Buffer is 2 10 fold for cell culture supernatants 20 fold for serum plasma and 30 fold cell tissue lysate Optimal sample dilution factor will depend on the abundance of target proteins If the background or antigen specific antibody signals are too strong the sample can be diluted further in subsequent experiments If the signal is too weak more concentrated samples can be used Completely remove Blocking Buffer from each well Add 400 ul of diluted samples into appropriate wells
11. d wash cells gently twice with cold 1X PBS taking care not to disturb cell layer li Add enough cold 1X PBS to cover cell layer and use cell scraper to detach cells Proceed to b Cells in Suspension b Cells in Suspension Pellet the cells by centrifuging using a microcentrifuge at 1500 rpm for 10 min 2 Make sure to remove any remaining PBS before adding 1X Cell Lysis Buffer 2X Cell Lysis Buffer should be diluted 2 fold with ddH20 Solubilize the cells at 2x10 cells ml in 1X Cell Lysis Buffer 3 Pipette up and down to resuspend cells and rock the lysates gently at 2 8 C for 30 minutes Transfer extracts to microfuge tubes and centrifuge at 13 000 rpm for 10 min at 2 8 C Note If the lysates appear to be cloudy transfer the lysates to a clean tube centrifuge again at 13 000 rpm for 20 minutes at 2 8 C If the lysates are still not clear store them at 20 C for 20 minutes Remove from the freezer and immediately centrifuge at 13 000 rpm for 20 minutes at 2 8 C 4 Transfer lysates to a clean tube Determining cell lysate concentrations using a total protein assay BCA Protein Assay Kit Pierce Prod 23227 Aliquot the lysates and store at 80 C 3 Extracting Protein from Crude Tissue 1 Transfer approximate 100 mg crude tissue into a tube with 1 ml 1X Cell Lysis Buffer 2X Cell Lysis Buffer should be diluted 2 fold with ddH20 RayBio L Series Human Antibody Array L 182 Protocol 9 2 Homogenize the tissue
12. fold in step 11 on page 11 b For labeling serum or plasma Add 22 ul of 1X Labeling Reagent Solution into a new tube containing 35 ul dialyzed serum or plasma sample and 155 ul Labeling Buffer Item K c For labeling cell or tissue lysates transfer 30 ug 15 ul of 2 mg ml cell or tissue lysates into a tube and add labeling buffer Item K for a total volume of 300 ul Then add 3 3 ul of 1X Labeling Reagent Solution RayBio L Series Human Antibody Array L 182 Protocol 10 Note To normalize serum plasma or cell tissue lysate concentrations during biotinylation measure sample volume before and after dialysis Then adjust the volumes of dialyzed serum plasma or cell tissue lysates and Labeling Buffer to compensate For example if the sample volume doubles after dialysis then use twice as much serum plasma in the labeling reaction 70 ul and reduce the Labeling Buffer to 120 ul 6 Incubate the reaction solution at RT with gentle rocking or shaking for 30 min Mix the reaction solution by gently tapping the tube every 5 minutes 7 Add 3 ul Stop Solution Item D into each reaction tube and immediately dialyze as directed in Steps 1 3 on pages 8 9 Note Biotinylated samples can be stored at 20 C or 80 C until you are ready to proceed with the assay C Drying the Glass Slide 8 Remove the package containing the Assembled Glass Slide Item E from the freezer Place unopened package on the bench top for approx 15 min and
13. hose steps lasting 2 hours or longer e During incubation and wash steps avoid foaming and remove all bubbles from the sub array surface e Perform all incubation and wash steps under gentle rotation or rocking motion 0 5 to 1 cycle sec e Wash steps in Wash Buffer Il and all incubation steps may be performed overnight at 4 C e Avoid cross contamination of samples to neighboring wells To remove Wash Buffers and other reagents from chamber wells you may invert the Glass Slide Assembly to decant and aspirate the remaining liquid RayBio L Series Human Antibody Array L 182 Protocol 7 e Unlike most Cy3 fluors the HiLyte Plus 555 used in this kit is very stable at room temperature RT and resistant to photobleaching on the hybridized glass slides However please protect glass slides from directly strong light and temperatures above RT IV Protocol Assay Diagram 1 Cell culture supernatants 2 Serum or plasma or cell tissue lysates 1 Serum sample x A I 1 Preparation of sample 4 2 Dialysis of sample 2 Dialysis of sample 3 Determination of protein concentration 3 Biotinylation of sample 4 Biotinylation of sample 4 Dialysis of biotinylated 5 Dialysis of biotinylated sample sample 5 proceed to microarray analysis 6 proceed to microarray analysis Note If using cell or tissue lysates start at Dialysis of sample RayBio L Series Human Antibody Array L 18
14. ibody Array L 182 Map 16 B RayBio Human Antibody Array L 182 Target List 16 VI Interpretation Of Results 17 A Explanation of Controls Spots 17 B Typical Results 18 C Background Subtraction 19 D Normalization of Array Data 19 E Threshold of Significant Difference 20 VII Troubleshooting Guide 21 VII Selected References 22 RayBio L Series Human Antibody Array L 182 Protocol l Introduction Obesity research has seen a surge in interest over the past 10 years One of the key driving forces is that adipose tissue is found no longer to be an inert energy storage organ but is emerging as an active participant in regulating physiological and pathologic processes Many soluble factors have been identified from the adipose tissue and are known as adipocytokines or adipokines Some adipokines such as leptin and resistin are produced mainly by the adipose tissues while others such as TNF alpha IL 6 MCP 1 and IL 1 are also synthesized in other tissues Because all of these factors can act in an autocrine paracrine or endocrine manner adipokines are thought to serve as mediators linking obesity inflammation immunity and other obesity related diseases Recent technological advances by RayBiotech have enabled the largest commercially available antibody array to date With the L Series Antibody Array 182 researchers can now obtain a broad panoramic view of adipokine expression The expression levels of 18
15. ion to automatically measure the local background around each spot For best results we recommend comparing signal intensities representing the MEDIAN background signals minus local background If your resulting fluorescence signal intensity reports do not include these values e g a column labeled as MED532 B532 you may need to subtract the background manually or change the default settings on your scanner s data report menu D Normalization of Array Data To normalize signal intensity data one sub array is defined as reference to which the other arrays are normalized This choice is arbitrary For example in our Analysis Tool Software described below the array represented by data entered in the left most column each worksheet is the default reference array You can calculate the normalized values as follows X Ny X y P1 P y Where P1 mean signal intensity of POS spots on reference array P y mean signal intensity of POS spots on Array y X y mean signal intensity for spot X on Array y X Ny normalized signal intensity for spot X on Array y The RayBio Analysis Tool software is available for use with data obtained using RayBio Biotin Label based Antibody Arrays You can copy and paste RayBio L Series Human Antibody Array L 182 Protocol 19 your signal intensity data with and without background into the Analysis Tool and it will automatically normalize signal intensities to the Po
16. ith endometriosis of different stages and those without endometriosi Biomarkers 2009 14 8 604 618 Yao Liang Tang et al Hypoxic Preconditioning Enhances the Benefit of Cardiac Progenitor Cell Therapy for Treatment of Myocardial Infarction by Inducing CXCR4 Circ Res 2009 109 197723 RayBio L series Antibody Arrays are patent pending technology developed by RayBiotech This product is intended for research only and is not to be used for clinical diagnosis Our produces may not be resold modified for resale or used to manufacture commercial products without written approval by RayBiotech Inc Under no circumstances shall RayBiotech be liable for any damages arising out of the use of the materials Products are guaranteed for six months from the date of shipment when handled and stored properly In the event of any defect in quality or merchantability RayBiotech s liability to buyer for any claim relating to products shall be limited to replacement or refund of the purchase price RayBio is a registered trademark of RayBiotech Inc HyLite Plus is a trademark of Anaspec Inc GenePix is a registered trademark of Molecular Devices Inc RayBio L Series Human Antibody Array L 182 Protocol 23 This product is for research use only 2011 RayBiotech Inc RayBio L Series Human Antibody Array L 182 Protocol 24
17. ly very close to background signals in each sub array RayBio L Series Human Antibody Array L 182 Protocol 17 B Typical Results The following figure shows the RayBio L Series Human Obesity Antibody Array 182 probed with serum sample The images were captured using a Axon GenePix laser scanner The strong signals in row 20 and the upper left and lower right corners of each array are Positive Controls which can be used to identify the orientation and help normalize the results between arrays RayBio L Series Human Obesity Antibody Array 182 Sample 1 Sample 2 If scanned using optimal settings 3 distinct signal intensities will be seen POS1 gt POS2 gt POS3 _ If all of these signals are of similar intensity try increasing or decreasing laser power and or signal gain settings NOTE in the absence of an external standard curve for each protein detected there is no means of assessing absolute or relative concentrations of different proteins in the same sample using immunoassays If you wish to obtain quantitative data ie concentrations of the various analytes in your samples try using our Quantibody Arrays instead RayBio L Series Human Antibody Array L 182 Protocol 18 C Background Subtraction Once you have obtained fluorescence intensity data you should subtract the background and normalize to the Positive Control signals before proceeding to analysis Most laser fluorescence scanner software have an opt
18. mple to a clean microfuge tube Spin dialyzed samples for 5 min at 10 000 rpm to remove any particulates or precipitates and then transfer the supernatants to a clean tube Note The sample volume may change during dialysis RayBio L Series Human Antibody Array L 182 Protocol 9 Note Dialysis procedure may proceed overnight Note Determine the total protein concentration for cell culture supernatants or cell tissue lysate after dialysis procedure Step 3 We recommended using a BCA total protein assay eg Pierce Catalog 23227 B Biotin labeling Sample Note Amines e g Tris glycine and azides quench the biotinylation reaction Avoid contaminating samples with these chemicals prior to biotinylation 4 Immediately before use prepare 1X Labeling Reagent Briefly spin down the Labeling Reagent tube Item B Add 100 ul 1X PBS into the tube then pipette up and down or vortex slightly to dissolve the lyophilized reagent 5 Add 1X Labeling Reagent to dialyzed samples a For labeling cell culture supernatants transfer 180 ul dialyzed sample into a new tube Add 36 ul of 1X Labeling Reagent Solution per 1 mg total protein in dialyzed cell culture supernatant Mix well For example if sample s total protein concentration is 0 5 mg ml you need to add 3 24 ul 1X Labeling Reagent to the tube of 180 ul dialyzed sample Note You need to biotin label 360 ul of dialyzed sample if dilution of the biotin labeled samples is 2
19. or 10 min Remove the wash buffer Repeat 2 times for a total of 3 washes 22 Repeat step 20 this time with 1X Wash Buffer II Repeat one time for a total of two washes for 5 min per wash 23 Finally wash the glass slide with 30 ml of ddH O for 5 min Remove glass slide and decant water from Centrifuge Tube 24 Remove buffer droplets from the slide completely by one of the following ways e Put the glass slide into the Slide Washer Dryer and dry the glass slide by centrifuge at 1 000 rpm for 3 minutes without cap e Or dry the glass slide by a compressed N2 stream e Or gently apply suction with a pipette to remove buffer droplets Do not touch the array only the sides Note Make sure the finished glass slide is completely dry before scanning or storage RayBio L Series Human Antibody Array L 182 Protocol 14 E Fluorescence Detection 25 You may proceed immediately to scanning or you may store the slide at 20 C in the Centrifuge Tube provided or at RT to scan at a later time Note Please protect the finished glass slides from temperatures above RT and store them in the dark Do not expose glass slide to strong light such as sunlight or a UV lamp Note If you need to repeat any of the incubation steps after finishing the experiment you must first re assemble the glass slide into the incubation chamber by following the steps as described below To avoid breaking the printed glass slide you may first want to pr
20. ow serum medium such as 0 2 FCS FBS serum and then incubate cells again for 48 hours The membrane based array is recommended if high serum medium such as 10 FCS FBS is used as high background can occur on glass slide arrays with high serum containing media samples To collect supernatants centrifuge at 1 000 g for 10 min and store as lt 1 ml aliquots at 80 C until needed Measure the total wet weight of cultured cells in the pellet and or culture dish You may then normalize between arrays by dividing fluorescent signals by total cell mass i e express results as the relative amount of protein expressed mg total cell mass Or you can normalize between arrays by determining cell lysate concentration using a total protein assay BCA Protein Assay Kit Pierce Prod 23227 The density of cells per dish used is dependent on the cell type More or less cells may be required Optimal culture time may vary and will depend on the cell line treatment conditions and other factors tBovine serum proteins produce detectable signals on the RayBio L Series Array in media containing serum concentrations as low as 0 2 When testing serum containing media we strongly recommend testing an uncultured media blank for comparison with sample results RayBio L Series Human Antibody Array L 182 Protocol 4 2 Extracting Protein from Cells 1 Centrifuge Cells a Adherent Cells i Remove supernatant from cell culture an
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22. r into the tube to prepare a concentrated Cy3 Conjugated Streptavidin stock solution Pipette up and down to mix gently do not store the stock solution for later use c To prepare 1X Cy3 Conjugated Streptavidin add 200 ul of the concentrated Cy3 Conjugated Streptavidin stock solution into a tube with 800 ul of Blocking Buffer Mix gently 18 Carefully remove Assembled Glass Slide from container Remove all of Wash Buffer II from the wells Add 400 ul of 1X Cy3 Conjugated Streptavidin to each sub array Cover the incubation chamber with the plastic adhesive strips Note Avoid exposure to light in Steps 19 25 by covering the Glass Slide Assembly with aluminum foil or incubate in a dark room 19 Incubate with 1X Cy3 Conjugated Streptavidin at RT for 2 hours with gentle rocking or shaking Note Incubation may be done overnight at 4 C RayBio L Series Human Antibody Array L 182 Protocol 13 20 Decant the solution and disassemble the glass slide from the incubation frame and chamber Disassemble the device by pushing clips outward from the side as shown below Carefully remove the glass slide from the gasket Note Be careful not to touch the printed surface of the glass slide which is on the pu gt 1 Omm J same side as the barcode 21 Gently place the glass slide into 30 ml Centrifuge Tube Item M Add enough 1X Wash Buffer to cover the entire glass slide about 30 ml Wash with gentle rocking or shaking f
23. re gently De gas solutions prior to use Prepare more reagent and completely cover arrays with solution Reagent evaporation Cover the incubation chamber with adhesive film during incubation Cross contamination from neighboring wells Avoid overflowing wash buffer between wells Comet tail formation Air dry the slide for at least 1 hour before usage General Inadequate detection Increase laser power so the highest standard concentration for each cytokine receives the highest possible reading yet remains unsaturated Overexposure Lower the laser power Dark spots Completely remove wash buffer in each wash step High Insufficient wash Increase wash time and use more wash buffer background Dust Minimize dust in work environment before starting experiment Slide is allowed to dry out Take additional precautions to prevent slides from dying out during experiment RayBio L Series Human Antibody Array L 182 Protocol 21 VIII Selected References Christina Scheel et all Paracrine and Autocrine Signals Induce and Maintain Mesenchymal and Stem Cell States in the Breast Cell 2011 145 926 940 Lin Y Huang R Chen L et al Profiling of cytokine expression by biotin labeled based protein arrays Proteomics 2003 3 1750 1757 Huang R Jiang W Yang J et al A Biotin Label based Antibody Array for High content Profiling of Protein Expression Cancer Genomics Proteomics 2010 7
24. sitive Controls To order the Analysis Tool please contact us at 1 770 729 2992 or info raybiotech com for more information E Threshold of Significant Difference After subtracting background signals and normalization to Positive Controls comparison of signal intensities between and among array images can be used to determine relative differences in expression levels of each protein between samples or groups Any 21 5 fold increase or lt 0 65 fold decrease in signal intensity for a single analyte between samples or groups may be considered a measurable and significant difference in expression provided that both sets of signals are well above background Mean background 2 standard deviations accuracy 95 RayBio L Series Human Antibody Array L 182 Protocol 20 VII Troubleshooting Guide Problem Cause Recommendation Inadeguate detection Increase laser power and PMT parameters Inadeguate reagent volumes or Check pipettes and ensure correct improper dilution preparation Short incubation time Ensure sufficient incubation time and Weak Signal change sample incubation step to overnight Too low protein concentration in sample Dilute starting sample less or concentrate sample Improper storage of kit Store kit as suggested temperature Don t freeze thaw the slide Uneven signal Bubble formed during incubation Arrays are not completed covered by reagent Handle and pipette solutions mo
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