ELISA
Contents
1. p38 MAPK Thr180 Tyr182 ELISA Kit Il HOW IT WORKS 1 Add cells 2 Treatment with stimulators 3 Fixing and blocking or inhibitors ee Li 4 Anti phospho protein antibody 5 HRP conjugated secondary 6 Develop with substrate or anti pan protein antibody antibody 1MB Color de Je k fe D LA L LA Fig 1 Cell Based protein phosphorylation procedure 3 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit Ill REAGENTS AND STORAGE Store entire kit at lt 20 C immediately upon arrival Kit must be used within the 6 month expiration date Avoid repeated freeze thaw cycles STORAGE AFTER ITEM COMPONENT 1 PLATE KIT 2 PLATE KIT INITIAL THAW A Uncoated 96 Well Microplate 1 plate 2 plates Room Temperature B 20X Wash Buffer A Concentrate 1vial 30 ml C 20X Wash Buffer B Concentrate 1vial 30 ml 28 C D Fixing Solution 1 vial 30 ml E 30XQuenching Buffer Concentrate 1vial 2 ml F 5X Blocking Buffer Concentrate 1vial 20 ml 2 8 C 1 month 1000X Mouse Anti phospho G Thr180 Tyr182 p38 MAPK 1 vial 7 ul 2 vials 7 ul ea Concentrate H EN E DAN PIS MAER 1vial 7 ul 2 vials 7 ul ea E 3 Cc ONE UNS J TMB Substrate 1 vial 12 ml 2 vials 12 ml ea 53 C K Stop Solution 1 vial 14 ml For up to 3 months unless otherwise stated or until expiration date Contains 0 2 M Sulfuric Acid IV ADDITIONAL MATERIALS REQU2. phospho gt amp A G Thr180 Tyr182 p38 MAPK S Concentrate e z H 1000X Mouse Anti p38 MAPK Wn o Ji Sa DR ae A ue yewo a 1X Blocking Buffer Mig i 2 solution ao 1000X HRP Conjugated Em ORE Anti Mouse IgG Concentrate oz ul lt x J TMB Substrate No Preparation N A K Stop Solution 5 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit VI ASSAY PROCEDURE NOTE ALL incubations and wash steps must be performed under gentle rocking or rotation 1 2 cycles sec 1 Design your experiment For example see Figure 2 below Anisomycin PA 0021 0021 0021 0021 0 min e GuecplQrerelaPuO CHO DC es Cp om OOO OCG KO RO O OU OT lU NO KENO U ms 200 COCOS OVC Os OC OX LOS NO POCO wmn 999 000 000 000 ut MONS uuo douce E E E E Anti Phospho p38 Anti p38 MAPK Inhibitor Anti Inhibitor 4 MAPK Phospho p38 Anti p38 MAPK Thr180 Tyr182 MAPK Thr180 Tyr182 Fig 2 Example of how to seed cells for RayBio cell based assay OPTIONAL If seeding HUVECs HMEC 1 or other loosely attached cells coat the Uncoated 96 Well Microplate ITEM A by adding 100 ul poly L Lysine Recommended Sigma Aldrich Cat P4832 into each well and then follow manufacturer s instructions A pre coated CellBIND microplate or other poly lysine treated tissue culture plate may be used in place of Item A 6 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit 2 Seed 100 L l o
3. 265 808 811 12 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit TROUBLESHOOTING GUIDE Problem Cause Solution 1 Low signal 1 Improper storage of 1 Store the kit according the ELISA kit to manual instructions Keep substrate solution in dark 2 Improper dilution 2 Ensure correct preparation of antibody and reagents 3 Cells drop off from 3 Some of treatments may the wells make cells drop off the wells Reduce inhibitor or activator concentration 2 High 1 Inadequate washing 1 Be sure to remove background all of washing solution and follow the recommendation for washing 2 Too much cells 2 Reduce the cell number 3 Large CV 1 Inaccurate pipetting 1 Check pipette 2 Remaining wash 2 Remove all of wash buffer in the well buffer 3 Cells drop off from 3 Please don t directly face the wells the cells with tips when adding reagents or wash buffer 13 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit NOTES 14 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit NOTES 15 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit NOTES 16 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit This product is for research use only 2004 RayBiotech Inc 17 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit
4. IRED 1 A model cell line protein tyrosine kinase inhibitors growth factors or cytokines Microplate reader capable of measuring absorbance at 450 nm 37 C incubator 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 Absorbent paper Distilled or deionized water Orbital shaker or oscillating rocker SEE UU ANI SON SUPE ae ae 4 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit V REAGENT PREPARATION NOTE Thaw all reagents to room temperature immediately before use If wash buffers contain visible crystals warm to room temperature and mix gently until dissolved NOTE Briefly centrifuge 1 000g ITEMS G H and before opening to ensure maximum recovery ITEM COMPONENT PREPARATION EXAMPLE A Uncoated 96 Well Microplate No Preparation N A B 20X Wash Buffer A Concentrate Dilute 20 fold with 25 mlof concentrate 475 ml of water C 20X Wash Buffar B Concentrate distilled or deionized water 500 ml of 1X working solution D Fixing Solution No Preparation N A Dilute 30 fold with 1mlofconcentrate 29 ml of wash buffer E 30X Quenching Buffer Concentrate 1X Wash Buffer A 30 mlof 1X workdngsclution Dilute 5 fold with 20 mlof concentrate 80 ml of water i PA BISenine BUN Pee ee DAN distilled or deionized water 100 ml of 1X working solution gt 1000X Mouse Anti
5. MARY 1 Seed 30 000 cells into each well and incubate overnight i 2 Apply various treatment inhibitors or activators according to manufacturer s instructions i 3 Add 100 ul of Fixing Solution into each well and incubate for 20 minutes at room temperature l 4 Add 200 ul of prepared 1X Quenching Buffer and incubate for 20 minutes at room temperature l 5 Add 200 ul of prepared 1X Blocking Buffer and incubate for 1 hour at 37 C 6 Add 50 ul of prepared 1X primary antibody to each well and incubate for 2 hours at room temperature 7 Add 50 ul of prepared 1X HRP Conjugated secondary antibody and incubate for 1 hour at room temperature l 8 Add 100 ul TMB Substrate and incubate 30 minutes at room temperature 9 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit 9 Add 50 ul Stop Solution to each well Read at 450 nm immediately VIII QUALITY CONTROL DATA Representative results of Cell Based p38 MAPK Thr180 Tyr182 are shown below 1 Seeded 100 ul 30 000 A431 cells into appropriate wells of the microplate Cells were incubated at 37 C in 5 CO overnight 2 Added 50 ul of different concentrations of stimulators rhEGF concentration for A431 cells 0 20 or 100 ng ml in serum free DMEM to appropriate wells shown below Then incubated for 10 20 or 30 min at 37 C 3 Discarded the solution and washed 3 times with 1X Wash Buffer A 200 ul each imm
6. SEW ej Cell Based Human Mouse Rat p38 MAPK Thr180 Tyr182 Phosphorylation ELISA Kit For the semi quantitative detection of phosphorylated human mouse or rat p38 MAPK Thr180 Tyr182 and total p38 MAPK in adherent whole cell lines User Manual Revised May 10 2013 Cat CBEL P38 1 1 plate kit Cat CBEL P38 2 2 plate kit Please read manual carefully before starting experiment RayBiotech Inc the protein array pioneer company Hil 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 Cell Based Human Mouse Rat p38 MAPK Thr180 Tyr182 Phosphorylation ELISA Kit TABLE OF CONTENTS OO asa eect UR ties 2 Il How lt Works ee sna nlne kanan ndina 3 Ill Reagents and Storage et 4 IV Additional Reagents Required 4 MV Reagent Pfreparal lol yyi eum B VI Assay Procedure nnn 6 VII Assay Procedure Summary 9 VIII Quality Control Data e 10 IX References EB BMDIMBDBIBIMIDMDMDMDMBMIMIMMDINMNaM 12 X Troubleshooting Guide ceti 13 1 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit I INTRODUCTION Protein phosphorylation is instrumental in the regulation of protein activity within a cell It plays important roles in the living cells including proliferation differentiation and metabolism A large number of protein kinases and phosphatases have been extensively investigated and have been
7. ediately Then flipped the microplate upside down and gently tapped to remove all of excess wash buffer The protocol was continued as stated C Anti Phospho p38 MAPK Thr180 Tyr182 ZZA Anti p38 C Anti Phospho p38 MAPK Thr180 Tyr182 Anti p38 MAPK 0 6 0 6 0 5 4 0 5 T B 0 4 E Z 0 3 bd 2 il n 03 a 02 0 2 9 0 1 0 1 0 0 0 0 Anisomycin 0 0 2 1 pg ml Anisomycin 0 0 2 1 ug ml concentrations concentrations Fig 3A Hela cells were stimulated by different Fig 3B Hela cells were stimulated by different concentrations of anisomycin for 15 minutes concentrations of anisomycin for 1 hour at 37 C at 37 C 10 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit Anisomycin 0 15 60 0 15 60 Min Anti p38 Anti p38 MAPK Thr180 182 Fig 4 Western blot analysis of extracts from 1 ug ml Anisomycin treated Hela cells Phospho p38 MAPK Thr180 Tyr182 and Anti p38 MAPK antibodies were used in both detection assays 11 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit IX REFERENCES 1 Winston B W et al 1997 J Immunol 159 4491 4497 2 Michael J Clemens and Michael C 1997 Protein Phosphorylation in Cell Growth Regulation 1 Edition Morinobu A et al 2002 PNAS 99 12281 12286 4 Han J et al 1994 Science
8. f 30 000 cells into each well of the Uncoated 96 Well Microplate ITEM A provided and incubate overnight at 37 C with 5 CO gt NOTE The optimal cell number used will vary on the cell line and the relative amount of protein phosphorylation More or less cells may be used but this must be determined empirically NOTE The cells can be starved 4 24 hours depending on cell line prior to treatment with inhibitors or activators 3 Apply various treatments inhibitors such as siRNA or chemicals or activators according to manufacturer s instructions and incubate for the desired time points NOTE It is recommended to dissolve inhibitors or activators into serum free cell culture medium before treating the cells unless otherwise stated in the manufacturer s instructions 4 Discard the cell culture medium by flipping the microplate upside down and gently tapping the bottom of the microplate over a sink 5 Wash by pipetting 200 ul of the prepared 1X Wash Buffer A ITEM B into each well Discard the wash buffer same as step 4 and wash 2 more times for a total of 3 washes using fresh wash buffer each time After the final wash gently blot the microplate onto a paper towel to remove any excess remaining buffer NOTE To avoid cell loss do not pipette directly onto the cells Instead gently dispense the liquid down the wall of cell culture wells Also avoid the use of vacuum suction or too forcefully tapping the microplate when disca
9. rding any solution 6 Add 100 ul of Fixing Solution ITEM D into each well and incubate for 20 minutes at room temperature NOTE The fixing solution is used to permeabilize the cells 7 Repeat wash step 5 7 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit 8 Add 200 ul of the prepared 1X Quenching Buffer ITEM E into each well and incubate 20 minutes at room temperature NOTE The quenching buffer is used to minimize the background response 9 Wash 4 times with 1X Wash Buffer A 10 Add 200 ul of the prepared 1X Blocking Buffer ITEM F into each well and incubate for 1 hour at 37 C 11 Wash 3 times with the prepared 1X Wash Buffer B ITEM C NOTE If needed the microplate may be stored at 80 C for several days after this wash 12 Add 50 ul of the prepared 1X primary antibody ITEM G or H into each corresponding well and incubate for 2 hours at room temperature 13 Wash 4 times with 1X Wash Buffer B 14 Add 50 ul of the prepared 1X HRP Conjugated secondary antibody ITEM I into each well and incubate for 1 hour at room temperature 15 Wash 4 times with 1X Wash Buffer B 16 Add 100 ul of the TMB Substrate ITEM J into each well and incubate for 30 minutes at room temperature in the dark 17 Add 50 ul of the Stop Solution ITEM K into each well Read at 450 nm immediately 8 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit VII ASSAY PROCEDURE SUM
10. shown to be involved in signal transduction pathways The RayBio Cell Based Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA kit is a very rapid convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in cells It can be used for measuring the relative amount of p38 MAPK Thr180 Tyr182 phosphorylation and screening the effects of various treatments inhibitors such as siRNA or chemicals or activators in cultured human mouse and rat cell lines By determining p38 MAPK protein phosphorylation in your experimental model system you can verify pathway activation in your cell lines without spending excess time and effort in preparing cell lysate and performing an analysis of Western Blot In the Cell Based p38 MAPK Thr180 Tyr182 ELISA kit cells are seeded into a 96 well tissue culture plate The cells are fixed after various treatments inhibitors or activators After blocking Anti Phospho p38 MAPK Thr180 Tyr182 or Anti p38 MAPK antibody is pipetted into the wells and incubated The wells are washed and HRP conjugated anti mouse IgG is added to the wells The wells are washed again a TMB substrate solution is added to the wells and color develops in proportion to the amount of protein The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm See Figure 1 below for an illustration 2 RayBio Human Mouse Rat Cell Based
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