Manual - RayBiotech, Inc.
Contents
1. HRP conjugated anti rabbit IgG concentrate should be diluted 1 000 fold with 1x Assay Diuent For example Briefly spin the vial ItemD and pipette up and down to mix gently Add 10 ul of HRP conjugated anti rabbit IgG concentrate into a tube with 10 ml 1x Assay Diluent to prepare a 1 000 fold diluted HRP conjugated anti rabbit IgG solution 7 Cell Lysate Buffer should be diluted 2 fold with deionized or distilled water before use recommend to add protease and phosphatase inhibitors VII ASSAY PROCEDURE 1 Bring all reagents to room temperature 18 25 C before use It is recommended that all samples or Positive Control should be run at least in duplicate Add 100 ul of each sample or positive control into appropriate wells see the following 96 well microplate formate Cover 7 RayBio Phospho P38 alpha MAPK Thr180 Tyr182 and pan P38 alpha MAPK ELISA Kit Protocol well with plate holder and incubate for 2 5 hours at room temperature or over night at 4 C with shaking 96 well microplate coated with phosphorylated and pan antibodies Anti p38 alpha MAPK Anti pan p38 MAPK Thr180 Tyr182 123 4 5 6 7 8 9 10 11 12 ITQOTmMmmMoOUOW Ly 2 Discard the solution and wash 4 times with 1x Wash Solution Wash by filling each well with Wash Buffer 300 ul using a multi channel pipette or autowasher Complete removal of liquid at each step is essential to good performance After the last wash remove any remai2. at room temperature 6 Add 50 ul Stop Solution to each well Read at 450 nm immediately IX TYPICAL DATA ELISA data analysis Average the duplicate readings for each sample or positive control i Positive Control Hela cells were treated with Anisomycin at 37 C for 10 min Solubilize cells at 4 x 10 cells ml in Cell Lysate Buffer Serial dilutions of lysates were analyzed in this ELISA Please see step 3 of Part VI Reagent Preparation for detail Assay Diluent 1 0 1 4 oN T T T T P 41 P 2 P 3 P 4 OD 450 nm Positive control dilution series 10 RayBio Phospho P38 alpha MAPK Thr180 Tyr182 and pan P38 alpha MAPK ELISA Kit Protocol i Anisomycin Stimulation of Hela Cell Lines Hela cells were treated or untreated with Anisomycin for 10 min at 37 C Cell lysates were analyzed using this phosphoELISA and Western Blot A ELISA 1 5 mE Untreated Hela Anisomycin treated Hela 1 0 4 c O lO q ll m O 05 0 0 p38 alpha MAPK Thr180 Tyr182 pan p38 alpha MAPK B Western Blot Analysis hEGF 10 0o 10 Min Anti phospho p38 alpha MAPK Anti pan p38 MAPK Thr180 Tyr182 11 RayBio Phospho P38 alpha MAPK Thr180 Tyr182 and pan P38 alpha MAPK ELISA Kit Protocol X TROUBLESHOOTING GUIDE secondary antibody dilution Problem Cause Solution 1 Sample signals a Too low a Sample concentration is a Increasing s
3. Cytokine Antibody Array Simultaneous detection up to 200 proteins cytokine chemokine growth factor adipokine angiogenic factor protease in one experiment Phosphorylation Antibody Array e RTK antibody array e EGFR phosphorylation antibody arrays Label based antibody array Simultaneous detection more than 500 proteins in one experiment Quantibody Array Quantitative measurement of multiple protein levels Protein Array ELISA Cell Based Phosphorylation ELISA Tissue MicroArray Protein Cytokine Chemokine Adiplokine Angiogenic factor Virus bacteria and infectious disease protein hormone Enzyme other Peptide Antibody Cytokine Adipokine Angiogenic factor Signal transduction Transcription factor Receptor Adhesion molecule Virus bacteria and other infectious agents Secondary antibody Tag antibody Immunoglobulin Hormone Cell surface Protease other 13 RayBio Phospho P38 alpha MAPK Thr180 Tyr182 and pan P38 alpha MAPK ELISA Kit Protocol Antibody array Protein array Peptide array ELISA Phosphorylation assay Tissue array Assay service just simply send your samples and get data in 1 to 2 weeks Antibody array Protein array ELISA Quantibody array Antibody production highest quality with very competitive price Monoclonal antibody Recombinant antibody Polyclonal antibody Phase display Antibody angineering Antibody conjugation Recombinant protein production Assay development Array printing Contact and non con
4. RayBio Phospho p38 alpha MAPK Thr180 Tyr182 and Pan p38 alpha MAPK ELISA Kit For Measuring Phospho p38 alpha MAPK Thr180 Tyr182 and Pan p38 alpha MAPK in Human and Mouse Cell Lysates User Manual Revised Feb 12 2013 RayBio Phospho p38 alpha MAPK Thr180 Tyr182 and Pan p38 alpha MAPK ELISA Kit Protocol Cat PEL P38 T180 T v RayBiotech Inc We Provide You With Excellent Protein Array System And Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 770 206 2393 Web www raybiotech com Email info raybiotech com RayBiotech Inc RayBio Phospho p38 alpha MAPK Thr180 Tyr182 and Pan p38 alpha ELISA Kit Protocol TABLE OF CONTENTS L FOC ore cees odvcnsectinencawicewrmenteendomanves 2 Il Material Provided ss cc icisccciscsevscetanuvaseadscvu 3 H 61 209 cee ee eee eer eee ee 3 IV Additional Materials Required 4 4 V Sample Preparation ccc cece eee eeee ees 4 VI Reagent Preparations ssciiccdetvadenviescnestlsleawaiecicnas 5 VII Assay PrOCEGUiG i ios icc nas oideecaeiveadesadicendcaeenss 7 VII Assay Procedure Summa ry 000ceeeees 9 IX Typical Dataiciccacccccceceee acon 10 ie Positive Control igi35 cieinacemeticrasnbriaseesionen 10 ii Anisomycin Stimulation of Hela Cell Lines 11 X Troubleshooting Guide cceeeeeee ee ees 12 1 RayBio Phospho P38 alpha MAPK Thr180 Tyr182 and pan P38 alpha MAPK ELISA K
5. ample too low concentration b Too high b Sample concentration is b Reducing sample too high concentration 2 Large CV a Inaccurate pipetting a Check pipettes 3 High background a Plate is insufficiently a Review the manual washed for proper washing If using an automated plate washer check that all ports are unobstructed b Contaminated wash b Make fresh wash buffer buffer 4 Positive Control a Improper storage of the a Upon receipt the kit Low signal ELISA kit should be stored at 20 C Store the positive control at 70 C after reconstitution b Stop solution b Stop solution should be added to each well before measurement and read OD immediately c Improper primary or c Ensure correct dilution 12 RayBio Phospho P38 alpha MAPK Thr180 Tyr182 and pan P38 alpha MAPK ELISA Kit Protocol RayBio ELISA kits Over 200 ELISA kits custom ELISA kit choose from over 500 list visit www raybiotech com for details RayBiotech Inc the protein array pioneer company strives to research and develop new products to meet demands of the biomedical community RayBio s patent pending technology allows detection of over 180 cytokines chemokines and other proteins in a single experiment Our format is simple sensitive reliable and cost effective Products include Cytokine Arrays Chemokine Arrays ELISA kits Phosphotyrosine kits Recombinant Proteins Antibodies and custom services Antibody Array
6. d down to resuspend and incubate the lysates with shaking at 2 8 C for 30 minutes 4 RayBio Phospho P38 alpha MAPK Thr180 Tyr182 and pan P38 alpha MAPK ELISA Kit Protocol Microcentrifuge at 13 000 rpm for 10 minutes at 2 8 C and transfer the supernates into a clean test tube Lysates should be used immediately or aliquoted and stored at 70 C Avoid repeated freeze thaw cycles Thawed lysates should be kept on ice prior to use For the initial experiment we recommend to do a serial dilution testing such as 5 fold and 100 fold dilution for your cell lysates with Assay Diluent Item E2 before use Note The fold dilution of sample used depends on the abundance of phosphorylated proteins and should be determined empiricallys More of the sample can be used if signals are too weak If signals are too strong the sample can be diluted further Cell Lysate Buffer should be diluted 2 fold with deionized or distilled water before use recommend to add protease and phosphatase inhibitors VI REAGENT PREPARATION 1 Bring all reagents and samples to room temperature 18 25 C before use 2 Item E2 Assay Diluent should be diluted 5 fold with deionized or distilled water before use 5 RayBio Phospho P38 alpha MAPK Thr180 Tyr182 and pan P38 alpha MAPK ELISA Kit Protocol 3 Preparation of Positive Control Briefly spin the Positive Control vial of Item K Add 400 ul 1x Assay Diluent Item E2 Assay Diluent sh
7. ed to the wells and color develops in proportion to the amount of p38 alpha MAPK Thr180 Tyr182 or pan p38 alpha MAPK bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm 2 RayBio Phospho P38 alpha MAPK Thr180 Tyr182 and pan P38 alpha MAPK ELISA Kit Protocol I MATERIAL PROVIDED 1 p38 alpha MAPK Microplate Item A 96 wells 12 strips x 8 wells coated with anti phospho P38 alpha MAPK Thr180 Tyr182 half plate red marker on left side and anti p38 alpha MAPK antibody half plate black marker on right side 2 Wash Buffer Concentrate 20x Item B 25 ml of 20x concentrated solution 3 Assay Diluent Item E2 15 ml of 5x concentrated buffer For diluting cell lysate sample detection antibody Item C and HRP conjugated Anti rabbit IgG Item D 1 4 Detection Antibody p38 alpha MAPK Item C 2 vial of rabbit anti p38 alpha MAPK each vial is enough to assay half microplate 5 HRP conjugated Anti rabbit IgG Item D 1 25 ul of 1 000x HRP conjugated anti rabbit IgG concentrate 6 TMB One Step Substrate Reagent Item H 12 ml of 3 3 5 5 tetramethylbenzidine TMB in buffered solution 7 Stop Solution Item I 8 ml of 0 2 M sulfuric acid 8 Cell Lysate Buffer Item J 5 ml 2x cell lysis buffer not including protease and phosphatase inhibitors 9 Positive Control HelaA003 1 Item K 2 vials of lyophilized powder from treated Hela cell lysa
8. it Protocol I INTRODUCTION RayBio Phospho p38 alpha MAPK Thr180 Tyr182 and Pan p38 alpha MAPK ELISA Enzyme Linked Immunosorbent Assay kit is a very rapid convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in cell lysates By determining phosphorylated p38 alpha MAPK protein in your experimental model system you can verify pathway activation in your cell lysates You can simultaneously measure numerous different cell lysates without spending excess time and effort in performing a Western Blot analysis This Sandwich ELISA kit is an in vitro enzyme linked immunosorbent assay for the measurement of human phospho p38 alpha MAPK Thr180 Tyr182 and pan p38 alpha MAPK help normalize the results of phospho p38 alpha MAPK from different cell lysate being compared An _ anti p38 alpha MAPK Thr180 Tyr182 half plate red marker on left side and anti pan p38 alpha MAPK antibody half plate black marker on right side has been coated onto a 96 well plate Samples are pipetted into the wells and phosphorylated left side and pan right side p38 alpha MAPK present in a sample is bound to the wells by the immobilized antibody The wells are washed and rabbit anti p38 alpha MAPK is used to detect phosphorylated or pan p38 alpha MAPK After washing away unbound antibody HRP conjugated anti rabbit IgG is pipetted to the wells The wells are again washed a TMB substrate solution is add
9. ning Wash Buffer by aspirating or decanting Invert the plate and blot it against clean paper towels 3 Add 100 ul of prepared 1x p38 alpha MAPK antibody Reagent Preparation step 5 to each well Incubate for 1 hour at room temperature with shaking 4 Discard the solution Repeat the wash as in step 3 8 RayBio Phospho P38 alpha MAPK Thr180 Tyr182 and pan P38 alpha MAPK ELISA Kit Protocol 5 Add 100 ul of prepared 1X HRP conjugated anti rabbit IgG solution see Reagent Preparation step 6 to each well Incubate for 2 hour at room temperature with shaking 6 Discard the solution Repeat the wash as in step 3 7 Add 100 ul of TMB One Step Substrate Reagent Item H to each well Incubate for 30 minutes at room temperature in the dark with shaking 8 Add 50 ul of Stop Solution Item I to each well Read at 450 nm immediately Vill ASSAY PROCEDURE SUMMARY 1 Prepare all reagents samples and standards as instructed 2 Add 100 ul sample or positive control to each well Incubate 2 5 hours at room temperature or over night at 4 C 3 Add 100 ul prepared 1X primary antibody to each well Incubate 1 0 hours at room temperature J 4 Add 100 ul prepared 1X HRP conjugated anti rabbit IgG solution Incubate 2 hour at room temperature RayBio Phospho P38 alpha MAPK Thr180 Tyr182 and pan P38 alpha MAPK ELISA Kit Protocol 9 5 Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes
10. ould be diluted 5 fold with deionized or distilled water before use into Item K vial to prepare a Positive Control P 1 Solution Dissolve the powder thoroughly by a gentle mix it can be removed by centrifuge if any precipitate in the solution is found See 1 Positive Control of part IX TYPICAL DATA for a typical result in page 9 Pipette 240 ul 1x Assay Diluent into each tube Use the Positive Control P 1 to produce a dilution series Shown below Mix each tube thoroughly before the next transfer 1x Assay Diluent serves as the background Vial of Item K 400 ul 1x Assay Diluent 120u 120 120 GTS OAT AT AN 2985 4 If the Wash Concentrate 20x Item B contains visible crystals warm to room temperature and mix gently until dissolved Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer 5 Briefly spin the detection antibody Item C before use Add 100 ul of 1x Assay Diluent into the vial to prepare a detection antibody concentrate Pipette up and down to mix 6 RayBio Phospho P38 alpha MAPK Thr180 Tyr182 and pan P38 alpha MAPK ELISA Kit Protocol gently the concentrate can be stored at 4 C for 5 days or at 80 C for one month The anti p38 alpha MAPK antibody should be diluted 55 fold with 1x Assay Diuent and used in step 4 of Part VII Assay Procedure 6 Briefly spin the HRP conjugated anti rabbit IgG Item D 1 before use Pipette up and down to mix gently
11. tact arrayers All kinds of substrates of your choice including glass slides membranes and plates 14 RayBio Phospho P38 alpha MAPK Thr180 Tyr182 and pan P38 alpha MAPK ELISA Kit Protocol 15 RayBio Phospho P38 alpha MAPK Thr180 Tyr182 and pan P38 alpha MAPK ELISA Kit Protocol 16 RayBio Phospho P38 alpha MAPK Thr180 Tyr182 and pan P38 alpha MAPK ELISA Kit Protocol This product is for research use only i 2004 RayBiotech Inc 17 RayBio Phospho P38 alpha MAPK Thr180 Tyr182 and pan P38 alpha MAPK ELISA Kit Protocol
12. te Hil STORAGE Upon receipt the kit should be stored at 20 C Please use within 6 months from the date of shipment After initial use Wash 3 RayBio Phospho P38 alpha MAPK Thr180 Tyr182 and pan P38 alpha MAPK ELISA Kit Protocol Buffer Concentrate Item B Assay Diluent Item E2 TMB One Step Substrate Reagent Item H Stop Solution Item I and Cell Lysate Buffer Item J should be stored at 4 C to avoid repeated freeze thaw cycles Return unused wells to the pouch containing desiccant pack reseal along entire edge and store at 20 C Item D 1 store at 2 8 C for up to one month store at 20 C for up to 6 months avoid repeated freeze thaw cycles Reconstituted Positive Control Item K should be stored at 70 C IV ADDITIONAL MATERIALS REQUIRED Microplate reader capable of measuring absorbance at 450 nm Protease and Phosphatase inhibitors Shaker Precision pipettes to deliver 2 ul to 1 ml volumes Adjustable 1 25 ml pipettes for reagent preparation 100 ml and 1 liter graduated cylinders Distilled or deionized water Tubes to prepare sample dilutions MDH AAIYNANB WN KR V SAMPLE PREPARATION Cell lysates Rinse cells with PBS making sure to remove any remaining PBS before adding the Cell Lysate Buffer Solubilize cells at 4 x 10 cells ml in 1x Cell Lysate Buffer we recommend adding protease and phosphatase inhibitors to Cell Lysate Buffer prior to sample preparation Pipette up an
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