LHO0001 - Protocol - Thermo Fisher Scientific
Contents
1. Add 100 uL of prepared 1X Goat Anti Rabbit IgG RPE to each well and incubate the plate for 30 minutes at room temperature on an orbital shaker Shaking should be sufficient to keep the beads suspended during incubation 500 600 rpm Prepare the Luminex 100 or 200 instrument during this incubation step Refer to the Luminex Instrument Quick Reference card provided in kit Refer to the INFORMATION SHEET for all bead regions and standard concentration values Remove the liquid from wells by aspiration with the vacuum manifold Wash beads by adding 200 uL Working Wash Solution to the wells allow the beads to soak for 10 seconds then aspirate with the vacuum manifold Repeat this washing step 2 additional times for a total of 3 washes Blot the bottom of the filter plate on clean paper towels to remove residual liquid Add 100 uL of Working Wash Solution to each well Shake the plate on an orbital shaker 500 600 rpm for 2 to 3 minutes to resuspend the beads Note If the plate cannot be read on the day of the assay cover and store the plate in the dark overnight at 2 to 8 C for reading the following day without significant loss of fluorescent intensity Aspirate Working Wash Solution from stored plates and add 100 uL fresh Working Wash Solution Place the plate on an orbital shaker for 2 to 3 minutes at 500 600 rpm prior to analysis Uncover the plate and insert the plate into the XY platform of the Luminex 100 or 200 instrume2. 26 iii Kit Contents and Storage Storage All components of the Akt Phospho 7 Plex Panel are shipped at 2 to 8 C Upon receipt store all kit components at 2 to 8 C Do not freeze Contents The components and amounts included in the Akt Phospho 7 Plex Panel are listed below Reagents Provided 100 Test Kit Akt Phospho 7 Plex Antibody Bead Concentrate 10X contains 0 25 mL x 1 vial 7 5 mM sodium azide Akt Phospho 7 Plex Standard 2 vial Akt Phospho 7 Plex Detector Antibody Concentrate 10X 0 50 mL x 1 vial contains 15 mM sodium azide Wash Solution Concentrate 20X contains 15 mM sodium azide 15 mL x 1 bottle Assay Diluent contains 15 mM sodium azide 15 mL x 1 bottle RPE Diluent contains 15 mM sodium azide 12 mL x 1 bottle Goat Anti Rabbit IgG RPE Concentrate 10X contains 15 mM 1 mL x 1 vial sodium azide Detector Antibody Diluent contains 3 3 mM thymol 12 mL x 1 bottle 96 well Filter Plate 1 x 96 well plate iv Overview Intended Use Background Information Introduction Invitrogen s Multiplex Bead Immunoassay Kits are developed to maximize flexibility in experimental design permitting the measurement of one or multiple proteins in panels designed by the researcher The Akt Phospho 7 Plex Panel contains a set of common reagents that are intended for use with the Luminex 100 or 200 dual laser detection system manufactured by Luminex Corporation and sold by Invitrogen and other vendors F
3. must be diluted at least 10 fold in Assay Diluent prior to analysis Lysate prepared using NP40 Lysis Buffer Cat no FNN0021 must be diluted at least 5 fold in Assay Diluent prior to analysis 2 Assays requiring Sample Treatment Buffer Lysate prepared using Cell Extraction Buffer Cat no FNN0011 must be diluted 2 fold with Sample Treatment Buffer and incubated for 20 minutes on ice Immediately after treatment the lysate must be diluted at least 5 fold in Assay Diluent with a net dilution of 10 fold Lysate prepared using NP40 Lysis Buffer Cat no FNN0021 must be diluted 2 fold with Sample Treatment Buffer and incubated for 20 minutes on ice Immediately after treatment the lysate must be diluted at least 4 fold in Assay Diluent with a net dilution of 8 fold 3 Samples with concentrations that exceed the standard curve should be diluted in Assay Diluent and reanalyzed Method of Washing Method of Washing Incomplete washing adversely affects assay results Perform all wash steps with the Wash Solution supplied with the kit All phases of the assay including incubations wash steps and loading beads are performed in the filter bottom plate supplied with the kit For a demonstration of proper washing techniques download Luminex video from www invitrogen com luminexinstrument l To wash beads place the filter plate on the vacuum manifold and aspirate the liquid with gentle vacuum do not exceed 5 mm Hg Excessive
4. sensitivity and quantitation of the assays are comparable to ELISAs Enzyme Linked Immuno Sorbent Assays Assay standards are calibrated to NIBSC National Institute for Biological Standards and Controls reference preparations when available to assure accurate and reliable results Continued on next page Overview Background Information Continued Assay Overview e 8 ex 0 Antibody Conjugated Beads x ec e lt gt Analyte Capture and Detection Antibody Analyte Detection Continued Invitrogen s Akt Phospho 7 Plex Panel is designed for the in vitro quantitative determination of Akt pS473 GSK 3p pS9 IGF IR pYpY1135 1136 IR pYpY1162 1163 IRS 1 pS312 p70S6K pTpS421 424 PRAS40 pT246 in cell lysates and tissue homogenates The antibodies used in this assay are human mouse and rat cross reactive This kit has not been tested for multiplexing with other markers Should user elect to multiplex this kit with other Luminex kits the assay conditions should be determined empirically for each specific application Visit the Invitrogen web site for a current listing of available Invitrogen multiplex bead immunoassays and reagents at www invitrogen com luminex The xMAP technology combines the efficiencies of multiplexing up to 100 different proteins for simultaneous analysis with reproducibility similar to ELISA The technology uses 5 6 um polystyrene beads which are internally dyed with red and in
5. the probe and reinstall Run an unclog protocol See instrument manual Readjust the instrument probe height If it is too low it could puncture the well membrane If it is too high air could be pulled up with the liquid which may appear as bead fragments to the instrument Continued on next page 19 Troubleshooting Continued Problem Cause Solution During washing The filter plate is clogged Dislodge the clog by gently pushing steps the vacuum the pointed end of a 15 mL plastic manifold does not conical tube into the bottom of the aspirate the liquid plate under the clogged well This from wells of the procedure clears the small opening filter plate in the plastic casing Dislodge by placing the filter plate on a clean paper towel and use a gloved thumb or a 1 mL Pasteur pipette bulb to plunge the top of the clogged well To prevent filter plate clogging clarify samples by centrifugation at 1 000 x g for 10 minutes prior to analysis Some samples may also require filtration prior to analysis Lack of a tight seal Hold the plate firmly against the vacuum manifold to form a tight seal If only a partial plate is being analyzed cover the empty wells with a self adhesive plate seal In house controls Incorrect concentration The standard proteins included in perform differently entered in data analysis Invitrogen s Antibody Bead Kits are in subsequent software calibrated to NIBSC preparations assays whenever possible T
6. the suggested reconstitution volume of Assay Diluent indicated on the INFORMATION SHEET Do not vortex When reconstituting protein solutions always avoid foaming 2 Replace the vial stopper and allow the vial to stand undisturbed for 10 minutes 3 Gently swirl and invert the vial 2 to 3 times to ensure complete reconstitution and allow the vial to sit at room temperature for an additional 5 minutes Preparing The standard curve is made by serially diluting the reconstituted Standard standard in Assay Diluent See below Do not vortex Mix by Curve gently pipetting up and down 5 to 10 times Transfer Volume 300 uL 300 uL S00pL 300pL 300yL 300pL Dilution N NNNN Blank e e e gt amp e e ESSE 18 1 16 1 32 1 164 Standard ux in mL Std1 Std2 Std3 Std4 Std5 Std6 Std7 Discard all remaining reconstituted and diluted standards after completing assay Return the Assay Diluent to the kit Continued on next page Preparing Reagents Continued M Sr Go to http www invitrogen com luminex under Multiplex Solution A Tools click Luminex Calculation Worksheet for auto calculation P of all assay dilutions Preparing 1X Determine the number of wells required for the assay Antibody Beads The Antibody Bead Concentrate is supplied as a 10X concentrate and must be diluted prior to use The fluorescent beads are light sensitive Protect antibody conjugated beads from light during handling 1 Immediate
7. vacuum can cause the membrane to tear resulting in antibody bead loss Prevent any vacuum surge by opening and adjusting the vacuum on the manifold before placing the plate on the manifold surface Stop the vacuum pressure as soon as the wells are empty Do not attempt to pull the plate off the vacuum manifold while the vacuum is still on or filter plate damage may occur Release the vacuum prior to removing the plate If solution remains in the wells during vacuum aspiration do not detach the bottom of the 96 well filter plate In some cases minor clogs in the filter plate may be dislodged by carefully pressing the bottom ofthe plate under the clogged well with the pointed end ofa 15 mL plastic conical tube Place the filter plate on a clean paper towel and use a gloved thumb or a 1 mL Pasteur pipette bulb to plunge the top of the clogged well Empty all clogged wells entirely before continuing the washes Note Do not attempt to repetitively pull vacuum on plates with clogged wells This can compromise the unclogged wells and bead loss may occur After all wells are empty lightly tap or press the filter plate onto clean paper towels hold the plate in the center for tapping to remove excess fluid from the bottom of the filter plate Do not invert plate Following the last aspiration and plate taps use a clean absorbent towel to blot the bottom of the filter plate before addition of next liquid phase or data acquisition step Do not
8. 7 Plex Panel according to instructions below Note bring all reagents and samples to room temperature before use Upon storage at 2 to 8 C a precipitate may form in the 20X Wash Solution Concentrate If this occurs warm the 20X Wash Solution Concentrate to 37 C and mix until the precipitate is dissolved l Prepare a 1X Working Wash Solution for use with a 96 well plate by transferring the entire contents of the Wash Solution Concentrate bottle to a 500 mL container or equivalent and then add 285 mL of deionized water Mix well The 1X Working Wash Solution is stable for up to 2 weeks when stored at 2 to 8 C Note To prepare smaller volumes of 1X Working Wash Solution mix 1 part of 20X concentrate with 19 parts of deionized water Mix well Each Kit comes with 2 complete sets of standard vials so that 2 runs on the plate can be made with freshly prepared standards Reconstitute the protein standard within 1 hour of performing the assay Additional standards are available from Invitrogen custom services Before performing serial dilutions confirm reconstitution volumes on the INFORMATION SHEET included in the Antibody Bead Kit s The concentrations of the protein components of the standard are indicated on the INFORMATION SHEET Perform standard dilutions in glass or polypropylene tubes Continued on next page Preparing Reagents Continued Reconstituting Lyophilized Standards 1 To the standard vial add
9. Plan Note Handle all blood components and biological materials as potentially hazardous Follow standard precautions as established by the Centers for Disease Control and Prevention and by the local Occupational Safety and Health Administration when handling and disposing of infectious agents This kit contains materials with small quantities of sodium azide Sodium azide reacts with lead and copper plumbing to form explosive metal azides Upon disposal flush drains with a large volume of water to prevent azide accumulation Avoid ingestion and contact with eyes skin and mucous membranes In case of contact rinse affected area with plenty of water Observe all federal state and local regulations for disposal It is recommended that a plate plan be designed before starting the assay A plate plan template is provided on page 26 for a fill in template The following is a suggested plate plan B blank Assay Diluent Standards 7 through 1 lowest concentration to highest The remainder of the plate is available for controls and samples which may be run as a singlet or in duplicate as desired Running all standards samples and controls in duplicate is recommended Preparing Reagents Introduction Preparing Wash Solution Guidelines for Standard Curve Preparation Review the information in this section before starting Prepare components of the Akt Phospho
10. Samples Introduction Cell Extraction Buffer Preparation This protocol has been applied to several human mouse and rat celllines Researchers should optimize the cell tissue extraction buffers and procedures for their own applications Recommended Cell Extraction Buffer Cell Extraction Buffer Invitrogen Cat no FNN0011 or 10 mM Tris pH 7 4 2 mM Na3VO 1 mM EDTA 100 mM NaCl 1 Triton X 100 1 mM EGTA 20 mM Na4P20 10 glycerol 1 mM NaF 0 5 deoxycholate 0 196 SDS Buffer without protease inhibitor cocktail and PMSF is stable for 2 3 weeks at 2 8 or 6 months when stored in aliquots at 20 C Add FRESH to the NP40 Lysis Bufer just before use e mM PMSF stock 0 3 M in DMSO e Protease inhibitor cocktail Sigma Cat no P 2714 An alternative cell extraction buffer is listed below Alternative Cell Lysis Buffer NP40 Lysis Buffer Invitrogen Cat no FNN0021 or 50 mM Tris pH 7 4 1 Nonidet P40 250 mM NaCl 5 mM EDTA 1 mM Na VO 50 mM NaF Buffer without protease inhibitor cocktail and PMSF is stable for 2 3 weeks at 2 8 or 6 months when stored in aliquots at 20 C Add FRESH to the NP40 Lysis Bufer just before use e mM PMSF stock 0 3 M in DMSO e Protease inhibitor cocktail Sigma Cat no P 2714 Continued on next page 11 Preparing Samples Continued Cell Lysis Procedure Important Sample Treatment Procedure 12 Non adherent cells Pellet cells by low speed centrifugation Remove m
11. al markers of disease status J Exp Med 200 331 343 Pickering A et al 2004 Cytokine response to infection with bacillus anthracis Spores Infect Immun 72 6382 6389 Piqueras B et al 2006 Upon viral exposure myeloid and plasmacytoid dendritic cells produce 3 waves of distinct chemokines to recruit immune effectors Blood 107 6 2613 2618 Raza K et al 2005 Early rheumatoid arthritis is characterized by a distinct and transient synovial fluid cytokine profile of T cell and stromal cell origin Arthritis Res amp Ther 7 4 R784 R795 Rice P et al 2005 Oral delivery and gastrointestinal absorption of soluble glucans stimulate increased resistance to infectious challenge J Pharmacol Exp Ther 314 3 1079 1086 Szodoray P et al 2004 Circulating cytokines in primary Sjorens Syndrome determined by a multiplex cytokine system Scand J Immunol 59 592 599 Talwar S et al 2006 Gene expression profiles of peripheral blood leukocytes after endotoxin challenge in humans Physiol Genomics 25 203 215 Wille Reece U et al 2004 Immunization with HIV 1 Gag protein conjugated to a TLR7 8 agonist results in the generation of HIV 1 Gag specific Th1 and CD8 T cell responses J Immunol 172 449 456 Williams D L et al 2005 Modulation of the phosphoinositide 3 kinase pathway alters innate resistance to polymicrobial sepsis J Immunol 174 7676 7683 Zacharowski K et al 2006 Toll
12. ditions You are also agreeing that the following terms and conditions constitute a legally valid and binding contract that is enforceable against you If you do not agree to all of the terms and conditions set forth below you must promptly return this Assay Product for a full refund prior to using it in any manner You the customer acquire the right under Luminex Corporation s patent rights if any to use this Assay Product or any portion of this Assay Product including without limitation the microsphere beads contained herein only with Luminex Corporation s laser based fluorescent analytical test instrumentation marketed under the name Luminex Instrument Invitrogen 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 Invitrogen warrants that all of its products will 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 Invitrogen Corporation s liability only to the cost 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 Invitrogen reserv
13. 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 website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 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 6500 Fax 813 5730 6519 Tech Fax 44 0 141 814 6117 E mail E mail et techsupport invitrogen com jpinfo invitrogen com eurotech invitrogen com Manufacturing Site MSDS Requests 22 Invitrogen Corporation 542 Flynn Road Camarillo CA 93012 USA Tel Toll Free 1 800 955 6288 E mail techsupport invitrogen com Material Safety Data Sheets MSDSs are available at www invitrogen com msds Purchaser Notification Limited Use Label License No 330 Luminex Assay Product Limited Warranty By opening the packaging containing this Assay Product which contains fluorescently labeled microsphere beads authorized by Luminex Corporation or using this Assay Product in any manner you are consenting and agreeing to be bound by the following terms and con
14. edium from the pellet and wash twice with ice cold PBS Remove the PBS and resuspend the cell pellet in cell lysis buffer recommended cell lysate concentration is 2 to 5 mg mL by gently pipetting Incubate 15 minutes on ice with occasional vortexing Transfer the lysate to a microfuge tube and centrifuge at 14 000 rpm for 10 minutes at 2 to 8 C Aliquot the cleared lysate into clean microfuge tubes and determine total protein concentration Adherent cells Remove tissue culture medium from the cells and wash twice with ice cold PBS Remove the PBS add cell lysis buffer recommended cell lysate concentration is 2 to 5 mg mL and incubate 15 minutes on ice Collect the cell lysate and transfer to a microfuge tube and centrifuge at 14 000 rpm for 10 minutes at 2 to 8 C Aliquot the cleared lysate into clean microfuge tubes and determine total protein concentration Storage Lysates should be frozen and stored at 80 C or analyzed shortly after collection Avoid multiple freeze thaw cycles of frozen samples Thaw completely mix well and clarify by centrifugation 14 000 rpm for 5 minutes prior to analysis to prevent clogging of the filter plates Certain samples may require incubation with Sample Treatment Buffer prior to analysis to improve recovery See analyte specific INFORMATION SHEET included with the Antibody Bead Kit 1 Assays not requiring Sample Treatment Buffer Lysate prepared using Cell Extraction Buffer Cat no FNN0011
15. es the right to select the method s used to analyze a product unless Invitrogen 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 Invitrogen 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 Invitrogen assumes 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 23 References The references below demonstrate the success customers achieve when using Invitrogen Multiplex Assays For complete list visit www invitrogen com luminex 10 11 Chang D H et al 2005 Sustained expansion of NKT cells and antigen specific T cells after injection of a galactosyl ceramide loaded mature dendritic cells in cancer patients J Exp Med 201 1503 1517 Kinter A et al 2004 CD25 CD4 regulatory T cells from the peripheral blood of asymptomatic HIV infected individuals regulate CD4 and CD8 HIV specific T cell immune responses in vitro and are associated with favorable clinic
16. frared fluorophores of differing intensities Each bead is given a unique number or bead region allowing differentiation of one bead from another Beads of defined spectral properties are conjugated to protein specific capture antibodies and added along with detector antibody samples including standards of known protein concentration control samples and test samples into the wells of a filter bottom microplate and where proteins bind to the capture antibodies and the protein specific detector antibodies bind to the appropriate immobilized proteins over the course of a 3 hour incubation After washing the beads R Phycoerythrin RPE conjugate is added and allowed to incubate for 30 minutes The RPE conjugate binds to the detector antibodies associated with the immune complexes on the beads forming a four member solid phase sandwich After washing to remove unbound RPE conjugate the beads are analyzed with the Luminex detection system By monitoring the spectral properties of the beads and the amount of associated RPE fluorescence the concentration of one or more proteins can be determined Experimental Overview Co Incubation Experimental outline for using the Akt Phospho 7 Plex Panel is Assay shown below Procedure Add beads Add detection antibody Add standards and samples then incubate for 3 hours Add RPE conjugate then incubate for 30 minutes Resuspend and acquire data using Luminex Detection system Me
17. handle higher speeds without splashing Ten to fifteen minutes prior to the end of the detector incubation step prepare the Goat Anti Rabbit IgG RPE and then proceed with Assay Reading Step 1 Continued on next page 15 Co Incubation Assay Procedure continued Preparing Goat The Goat Anti Rabbit IgG RPE is supplied as a 10X concentrate Anti Rabbit and must be diluted prior to use Protect Goat Anti Rabbit IgG RPE IgG RPE from light during handling To prepare a 1X Goat Anti Rabbit IgG RPE stock dilute 10 uL of 10X Goat Anti Rabbit IgG RPE in 100 of uL RPE Diluent per assay well Each well requires 100 uL of the diluted Goat Anti Rabbit IgG RPE See table below for examples of volumes to combine Note Dilution factor is 1 11 for extra pipetting volume Number of Vol 10X Vol RPE Wells Goat Anti Rabbit IgG RPE Diluent Concentrate 24 0 24 mL 2 4 mL 32 0 32 mL 3 2 mL 40 0 40 mL 4 0 mL 48 0 48 mL 4 8 mL 56 0 56 mL 5 6 mL 64 0 64 mL 6 4 mL 72 0 72 mL 7 2 mL 80 0 80 mL 8 0 mL 88 0 88 mL 8 8 mL 96 0 96 mL 9 6 mL Continued on next page 16 Co Incubation Assay Procedure continued Assay Reading 10 11 Remove the liquid from wells by aspiration with the vacuum manifold Add 200 uL Working Wash Solution to the wells Allow the beads to soak for 15 to 30 seconds then aspirate with the vacuum manifold Repeat this washing step Blot the bottom of the filter plate on clean paper towels to remove residual liquid
18. his calibration assures lot to lot consistency in performance However the concentration of the reconstituted standards may vary with each new lot of standard Therefore it is important to check the concentration of the standard listed on the INFORMATION SHEET and to verify all concentration values entered into the data analysis software Improper reconstitution or Check standard reconstitution and dilution ofthe standard dilution as described on page 8 Continued on next page 20 Troubleshooting Continued Problem Leaky plate Cause Solution remains on the bottom of the wells after vacuum aspiration causing wicking and leakage of well contents during next incubation Filter plate membrane tearing Solution After final wash step and plate taps use a clean absorbent towel to blot the bottom of the filter plate before addition of next liquid phase or data acquisition step Excessive vacuum can cause the membrane to tear resulting in antibody bead loss Prevent any vacuum surge by opening and adjusting the vacuum on the manifold before placing the plate on the manifold surface 21 Appendix Technical Support World Wide Visit the Invitrogen website at www invitrogen com for Web e Technical resources including manuals INFORMATION SHEET quick calculation worksheet application notes MSDSs FAQs formulations citations handbooks and more e Complete Technical Support contact information
19. ik Akt Phospho 7 Plex Panel For simultaneous quantitative determination of Akt pS473 GSK 3 pS9 IGF 1R pYpY1135 1136 IR pYpY1162 1163 IRS 1 pS312 p70S6K pTpS421 424 PRAS40 pT246 in human mouse or rat cell lysates and tissue homogenates Catalog no LHO0001 Rev 0 0 19 December 2008 PRLHO0001 Table of Contents Tableof Contents achte elle ep Bade ill Kit Contents and Storage iere e Here ree e e ERRARE Mee iv MAELO CEL ON uia oor herus een 1 OVETVIE WA ie er UH RR te Tried eee etti der Weg iore eset isa 1 L iiip ro ecL 4 Before Starting oe AeA ee aA RI AEE GG GSE ne 4 Preparing Reagents iie iere je itia tnde eren iere rra enata eee ie aene de earns 7 Preparing Samples eerte aire eed eee Eae eon bonn oe uu ep de Deed Ede dias 11 Method of Washing ccceccseesceseceseceeseceseesecaaecseesecaecaaecaececeseceaecaeeeeeeaeenaeeneees 13 Co Incubation Assay Procedure cccscessceseceseeseeseceseceecaecseeeeceaeeaaecaeeeeeesecnaeeneees 15 Performance Characteristics and Limitations of the Procedure 18 Troubl sShOOtlfg ze ee er a E US rennen 19 Desde 22 Technical Supports 5 c tert ede cd cb e ines 22 Putrch serNOtfICAtIOID iecore rtt e ER IR Ee RE Rea DES ede ES a 23 References d ee eS ed D Ad ses 24 Co Incubation Protocol Summary esee eene 25 Plate Plan Template e Robb en edhereed v dbaidhakenqenag v
20. leave plate on absorbent surface when adding reagents Continued on next page 13 Method of Washing Continued 1472 Reverse pipetting recommendation MM d To reduce bubbles and loss of reagents due to residual fluid left in 9 pipette tips use the recommended reverse pipetting technique E gt 1 To reverse pipette set the pipette to the appropriate volume needed Note Do not reverse pipette volumes 20 uL 2 Press the push button slowly to the first stop and then press on past it Note the amount past the first stop will depend on the volume of liquid available to aspirate from 3 Immerse the tip into the liquid just below the meniscus Release the push button slowly and smoothly to the top resting position to aspirate the set volume of liquid 5 Place the end of the tip against the inside wall of the recipient vessel at an angle 6 Press the push button slowly and smoothly to the first stop Some liquid will remain in the tip this should not be dispensed 7 Remove the tip keeping the pipette pressed to the first stop Note Bring all reagents and samples to room temperature before use 14 Co Incubation Assay Procedure Analyte Capture and Detection Use an adhesive plate cover to seal any unused wells This will keep the wells dry for future use Pre wet the designated assay wells by adding 200 uL of Working Wash Solution into designated wells Incubate plate 15 to 30 seconds at room tempe
21. like receptor 4 plays a crucial role in the immune adrenal response to systemic inflammatory response syndrome Proc Natl Acad Sci USA 103 16 6392 6397 2008 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 24 Co Incubation Protocol Summary Pre wet plate Add 25 uL 1X antibody coated beads and 200 uL Wash Solution Wash 1 x 200 uL Add 50 uL 1X detector antibody Sample type Standard Cell lysate Add 50 uL Add 50 uL standard appropriately diluted sample Shake for 3 hr at RT in the dark Wash 2 x 200 uL Add 100 uL 1X anti Rabbit RPE Shake for 30 min at RT in the dark Wash 3x 200 uL Add 100 uL wash buffer Shake for 2 3 min Read in Luminex Detection System Total time 3 5 hr Detector R phycoerythrin antibody RPE 25 Plate Plan Template aeq Jequinw 101 qI eld Jequunu ojee Uy 26 invitrogen Corporate Headquarters en Corporation n Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country ific contact information visit our web site at www invitrogen com
22. ly before dispensing vortex the 10X Antibody Bead Concentrate for 30 seconds followed by sonication in a sonicating water bath for 30 seconds 2 Prepare 1X Antibody Bead stock by diluting 2 5 uL of 10X beads in 25 uL of Working Wash Solution page 7 per assay well Each well requires 25 uL of the diluted beads See table below for examples of volumes to combine Note Dilution factor is 1 11 for extra pipetting volume Number of Wells Vol 10X Antibody Bead Vol Working Wash Concentrate Solution 24 0 06 mL 0 6 mL 32 0 08 mL 0 8 mL 40 0 10 mL 1 0 mL 48 0 12 mL 1 2 mL 56 0 14 mL 1 4 mL 64 0 16 mL 1 6 mL 72 0 18 mL 1 8 mL 80 0 20 mL 2 0 mL 88 0 22 mL 2 2mL 96 0 24 mL 2 4 mL Continued on next page Preparing Reagents Continued Preparing 1X The Detector Antibody is supplied as a 10X concentrate and Detector must be diluted prior to use Antibody To prepare a 1X Detector Antibody stock dilute 5 uL of 10X Detector Antibody in 50 uL of Detector Antibody Diluent per assay well Each well requires 50 uL of the diluted Detector Antibody See table below for examples of volumes to combine Note Dilution factor is 1 11 for extra pipetting volume Number of Wells Vol 10X Detector Vol Detector Antibody Concentrate Antibody Diluent 24 0 12 mL 1 2 mL 32 0 16 mL 1 6 mL 40 0 20 mL 2 0 mL 48 0 24 mL 24mL 56 0 28 mL 2 8 mL 64 0 32 mL 32mL 12 0 36 mL 3 6 mL 80 0 40 mL 4 0 mL 88 0 44 mL 44 mL 96 0 48 mL 4 8 mL 10 Preparing
23. mperature water bath for 20 minutes except plate and standard vials The fluorescent beads are light sensitive Protect the beads from light to avoid photobleaching of the embedded dye Use aluminum foil to cover test tubes used in the assay Cover filter plates containing beads with an opaque or aluminum foil wrapped plate cover Since the amber vial does not provide full protection keep the vial covered in the box or drawer when not in use Do not expose beads to organic solvents Do not place filter plates on absorbent paper towels during loading or incubations as liquid may be lost due to contact wicking An extra plate cover is a recommended surface to rest the filter plate Following plate washing remove excess liquid and blot from the bottom of the plate by pressing the plate on clean paper towels When pipetting reagents maintain a consistent order of addition from well to well to ensure equal incubation times for all wells To prevent filter tearing avoid touching the filter plate membrane with pipette tips Do not use reagents after kit expiration date It is recommended that in house controls be included with every assay If control values fall outside pre established ranges the assay may be suspect Contact Invitrogen Technical Support for product and technical assistance Do not mix or substitute reagents with those from other lots or sources Continued on next page Before Starting Continued Recommended Plate
24. nt and analyze the samples Determine the concentration of samples from the standard curve using curve fitting software It is recommended to use the five parameter algorithm with a weighted function 1 y depending on the software package used 17 Performance Characteristics and Limitations of the Procedure Performance Characteristics Procedure Limitations 18 Refer to analyte specific INFORMATION SHEET for performance claims Do not extrapolate the standard curve beyond the highest or lowest standard point the dose response and data collected in these regions may be non linear and should be considered inaccurate Note In some cases further dilution of the standard beyond 7 points may be possible to extend the low end of the standard curve Dilute samples that are greater than the highest standard with Assay Diluent or appropriate matrix diluent reanalyze these samples and multiply results by the appropriate dilution factor Samples are diluted in the assay be sure to account for this dilution factor during sample calculations The influence of various drugs aberrant sera hemolyzed hyperlipidemic jaundiced etc and the use of biological fluids in place of serum plasma and tissue culture supernatant samples have not been thoroughly investigated The rate of degradation of analytes in various matrices may not have been investigated The immunoassay literature contains frequent references to aberrant signals
25. or research use only Not intended for any animal or human therapeutic or diagnostic use Advances in the field of cell biology have defined a complex and interdependent set of extracellular and intracellular signaling molecules that control normal cell function Perturbations in signaling pathways may be important indicators and possibly the root cause of many diseases Therefore there is growing interest among clinicians as well as drug discovery groups in simultaneously monitoring multiple components of signaling pathways Solid phase multiplex protein assays are the tools of choice in these studies as they maximize efficiency by simultaneously profiling several proteins within individual samples Invitrogen s Multiplex Bead Immunoassays are solid phase protein immunoassays that use spectrally encoded antibody conjugated beads as the solid support The spectral beads are suitable for use in singleplex assays or may be mixed for multiplex assays according to the researcher s requirements Each assay is carefully designed and tested to assure that sensitivity range and correlation are maximized The assay is performed in a 96 well plate format and analyzed with a Luminex 100 or 200 instrument which monitors the spectral properties of the capture beads while simultaneously measuring the quantity of associated fluorophore Standard curves generated with this assay system extend over several orders of magnitude of concentrations while the
26. rature Aspirate the Working Wash Solution from the wells using the vacuum manifold Vortex the diluted bead solution prepared on page 9 for 30 seconds then sonicate for at least 30 seconds immediately prior to use in the assay Pipette 25 uL of the diluted bead solution into each well Once the beads are added to the plate keep the plate protected from light Add 200 uL Working Wash Solution to the wells Allow the beads to soak for 15 to 30 seconds Aspirate the Working Wash Solution from the wells with the vacuum manifold Repeat this washing step Blot the bottom of the filter plate on clean paper towels to remove any residual liquid Note Place the filter plate on a plate cover or non absorbent surface before all incubations 9 10 11 12 13 Add 50 uL of prepared 1X Detector Antibody page 10 to the wells To wells designated for the standard curve pipette 50 uL of appropriate standard dilution To the wells designated for the sample pipette 50 uL of sample Suggested total protein per well 10 to 40 ug However the exact amount should be determined by the individual user Cover filter plate containing beads with an aluminum foil wrapped plate cover Incubate the plate for 3 hours at room temperature on an orbital shaker Shaking should be sufficient to keep beads suspended during the incubation 500 600 rpm Larger radius shakers will need a lower speed and smaller radius shakers will typically
27. seen with some sera attributed to heterophilic antibodies Though such samples have not been seen to date the possibility of this occurrence cannot be excluded Troubleshooting Introduction Refer to the table below to troubleshoot problems encountered with the use of Invitrogen s Multiplex Bead Kits on the Luminex platform To troubleshoot problems with the Luminex instrument refer to the manual supplied with the instrument For more troubleshooting solutions visit www invitrogen com luminex Problem Cause During data Bead aggregation analysis insufficient and or erratic bead count is observed Loss of beads due to the filter plate membrane tearing Clog in instrument or probe Probe height set incorrectly Solution Make sure to vortex the beads for 30 seconds and then sonicate the beads for at least 30 seconds prior to beginning the assay to break up any bead aggregates Empty wells and add fresh wash buffer Shake for 2 to 3 minutes to resuspend the beads To prevent membrane tearing place pipette tips on the side of the well rather than straight down onto the membrane when dispensing liquid into the wells Turn the vacuum manifold on before placing the filter plate on the top to prevent vacuum surge When evaluating a new vacuum manifold adjust the vacuum force so that 3 seconds are required to empty 0 2 mL from the wells of a plate Remove probe sonicate for 5 minutes rinse
28. thods Before Starting Materials Required but Not Provided Luminex xMAP system with data acquisition and analysis software Invitrogen Cat no MAP0200 contact Invitrogen for instrument and software placement services see page 22 Filtration vacuum manifold for bead washing Pall Cat no 5017 is recommended Sonicating water bath Vortex mixer Orbital shaker small diameter rotation recommended Calibrated adjustable precision pipettes preferably with disposable plastic tips A manifold multi channel pipette is desirable Distilled or deionized water Glass or polypropylene tubes Aluminum foil or opaque 96 well plate cover Invitrogen Cat no PC10 Continued on next page Before Starting Continued Note Review the procedural notes below before starting the protocol This kit has not been tested for multiplexing with other markers Should the user elect to multiplex this kit with other Luminex kit the assay conditions should be determined empirically for each specific application Do not invert the filter plates during the assay The filter plates are designed to be used in conjunction with a vacuum manifold do not exceed 5 mm Hg and emptied from the bottom Do not freeze any component of this kit Store kit components at 2 to 8 C when not in use Allow all reagents to warm to room temperature before use air warm all reagents at room temperature for at least 30 minutes or alternatively in a room te
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