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1. 20 or 30 min at 37 C 3 Discarded the solution and washed 3 times with 1X Wash Buffer A 200 ul each immediately Then flipped the microplate upside down and gently tapped to remove all of excess wash buffer The protocol was continued as stated Anti Phospho p38 MAPK Thr180 Tyr182 ZA Anti p38 EZ Anti Phospho p38 MAPK Thr180 Tyr182 Anti p38 MAPK 0 6 0 5 0 4 E 0 4 2 g 03 Z 03 i ji S o 0 2 d 0 1 0 1 0 0 0 0 Anisomycin 0 0 2 1 ug 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 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 Ed2. 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 p38 MAPK Thr180 Ty
3. 00 ml of 1Xworking solution z 1000X Mouse Anti phospho amp A G Thr180 Tyr182 p38 MAPK Ss Concentrate 2 H 1000X Mouse Anti p38 MAPK T d Concentrate Dilute 1000 fold with TOH OEE t PPO L ek Blocking buffer 10 ml of 1X working gt 1X Blocking Buffer A solution g Q 1000X HRP Conjugated OE Anti Mouse IgG Concentrate oz 2 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 hea 0021 0021 0021 0021 0 min va DS OEC KOK O OO FORO RO NOS omn OOO l le NOOO Oe OOO Me ELO OPEM wmn OOO OO OO Gi Or COG KOR OO OTO TO O wn 999 999 999 999 m ROCNOIOQGIEU Eme T e E E E Anti Phospho p38 Anti p38 MAPK Inhibitor Anti Inhibitor MAPK Phospho p38 Anti p38 MAPK Thr180 Tyr182 MAPK Thr180 Tyr182 Fig 2 Example of plate layout 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 p
4. RayBio 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 Sept 29 2014 Cat CBEL P38 1 1 plate kit Cat CBEL P38 2 2 plate kit Cat CBEL P38 5 5 plate kit Please read manual carefully before starting experiment Ca RayBiotech Inc vi the protein array pioneer company 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 k introduction ae eee tse eae 2 ll HOw WOT C 3 I Reagents and Storage 4 IV Additional Reagents Required 4 V RespentPreparallolll susy Itu DEMENS 5 VI Assay Procedure nn 6 VII Assay Procedure Summary 9 VIII Quality Control Data nnn 10 IX References n 12 X Troubleshooting Guide nn 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 shown to be involved
5. cell culture wells Also avoid the use of vacuum suction or too forcefully tapping the microplate when discarding 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 immediatel
6. io Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit
7. ition Morinobu A et al 2002 PNAS 99 12281 12286 4 Han J et al 1994 Science 265 808 811 ed 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 RayB
8. lace of Item A 6 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit 2 Seed 100 ul of 30 000 cells into each well of the Uncoated 96 Well Microplate ITEM A provided and incubate overnight at 37 C with 5 CO 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
9. r182 ELISA Kit Il HOW IT WORKS 1 Add cells ee 4 Anti phospho protein antibody or anti pan protein antibody a 3 Fixing and blocking 6 Develop with substrate 2 Treatment with stimulators or inhibitors HA 5 HRP conjugated secondary antibody Je Je i TMB Color r 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 1plate 2 plates Room Temperature B 20X Wash Buffer A Concentrate 1 vial 30 ml C 20X Wash Buffer B Concentrate 1 vial 30 ml 28 C D Fixing Solution 1 vial 30 ml E 30X Quenching Buffer Concentrate 1 vial 2 ml F 5X Blocking Buffer Concentrate 1 vial 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 uH keya ARES MARK 1vial 7 ul 2 vials 7 j l ea sd 1000X HRP Conjugated Anti Mouse L ce PI TA HOH 2 vials 10 ul ea J TMB Substrate 1 vial 12 ml 2 vials 12 ml ea due K Stop Solution 1 vial 14 ml For up to 3 months unless otherwise stated or until expira
10. tion date Contains 0 2 M Sulfuric Acid IV ADDITIONAL MATERIALS REQUIRED 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 wed ee u 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 I 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 Buffer B Concentrate distilled or deionized water 500 ml of 1Xworking solution D Fixing Solution No Preparation N A Dilute 30 fold with 1mlof concentrate 29 ml of wash buffer E sex QUENENINE BYST Concentrate 1X Wash Buffer A 30mlof 1X working solution Dilute 5 fold with 20 ml of concentrate 80 ml of water j FA Pocking Bulle Concentrate distilled or deionized water 1
11. y 8 RayBio Human Mouse Rat Cell Based p38 MAPK Thr180 Tyr182 ELISA Kit VII ASSAY PROCEDURE SUMMARY 1 Seed 30 000 cells into each well and incubate overnight l 2 Apply various treatment inhibitors or activators according to manufacturer s instructions l 3 Add 100 ul of Fixing Solution into each well and incubate for 20 minutes at room temperature J 4 Add 200 ul of prepared 1X Quenching Buffer and incubate for 20 minutes at room temperature l 5 Add 200 L l of prepared 1X Blocking Buffer and incubate for 1 hour at 37 C J 6 Add 50 ul of prepared 1X primary antibody to each well and incubate for 2 hours at room temperature l 7 Add 50 L l of prepared 1X HRP Conjugated secondary antibody and incubate for 1 hour at room temperature J 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 l 9 Add 50 L l 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 Hela 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 Anisomycin Anysomycin concentration for Hela cells 0 0 2 or 1 ug ml in serum free DMEM to appropriate wells shown below Then incubated for 10
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