Please read manual carefully before starting
Contents
1. 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 1 G 2 G 3 H 1 H 2 or H 3 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 l 2 into each well and incubate for 1 hour at room temperature 15 Repeat step 13 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 Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Kit Protocol Vil ASSAY PROCEDURE SUMMARY 1 Seed 10 000 to 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 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 l 6 Add 50 L l of prepared 1X primary antibody into each well and inc2. Mouse IgG Concentrate 2 vials 10 L l ea J TMB Substrate 2 vials 12 ml ea T K Stop Solution 1 vial 14 ml 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 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 00 00 OTB er a 4 RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Kit Protocol 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 TEM COMPONENT PREPARATION EXAMPLE A Uncoated 96 Well Microplate No Preparation N A 20X Wash Buffer A Concentrate Dilute 20 fold with distilled or deionized 25 ml of concentrate 475 ml of water 20X Wash Buffer B Concentrate Wale SOO miot Tx working solution D Fixing Solu
3. C Anti p38 MAPK Thr180 182 Fig 4 3 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 13 RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Kit Protocol REFERENCES Winston B W et al 1997 J Immunol 159 4491 4497 Michael J Clemens and Michael C 1997 Protein Phosphorylation in Cell Growth Regulation 1 Edition Clark E A amp Hynes R O 1996 J Biol Chem 271 14814 14 RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Kit Protocol X 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 buffe
4. GF ng ml O 20 100 O 20 100 O 20 100 O 20 100 O O O O O O O e O O O O 0 min 10 min GOOO OCOL Z e 30 min OOOO OO WOOO OOO SOOO OOO ONO OO QOD OOO POO OOD COO OO OOOO Oo OOOO OOO SAO SIO Anti Phospho Anti Pan Inhibitor Inhibitor Specific Ab Specific Ab Anti Phospho Anti Pan Specific Ab Specific Ab 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 place of ITEM A 2 Seed 100 ul of 10 000 to 30 000 cells into each well of the Uncoated 96 Well Microplate ITEM A provided and incubate overnight at 37 C with 5 CO3 6 RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Kit Protocol 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 a
5. RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK Phosphorylation ELISA Sampler Kit For the semi quantitative detection of phosphorylated human mouse or rat ERK1 2 Thr202 Tyr204 JNK Thr183 Tyr185 p38 MAPK Thr180 Tyr182 and total ERK1 2 JNK p38 MAPK in adherent whole cell lines User Manual Revised July 16 2015 Cat CBEL ERK SK 2 plate kit Please read manual carefully before starting experiment RayBiotech Inc fie 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 ERK1 2 JNK p38 MAPK Phosphorylation ELISA Sampler Kit TABLE OF CONTENTS l PCPOCUCTION EM MD D IDDMDDBMDBADJ JMDMDJMDMMMMNa 2 Ne TOWN OS oer EEDEIEIEDEMDNRDRDITPEDIeEuPI EDE DE E 3 I Reagents and Storage 4 IV Additional Reagents Required 4 V Reagent Preparation ennn 5 VI Assay Procedure n 6 VII Assay Procedure SUMMary n 9 VII Quality Control Data icc cece eee yeas aera 10 IX References nnn 14 X Troubleshooting Guide n 15 1 RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Kit Protocol 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 phosphat
6. ases have been extensively investigated and have been shown to be involved in signal transduction pathways The RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Sampler 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 ERK1 2 Thr202 Tyr204 JNK Thr183 Tyr185 and 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 specific 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 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 protein specific antibody or anti pan protein specific 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 b
7. elow for an illustration 2 RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Kit Protocol ll HOW IT WORKS 1 Add cells 2 Treatment with stimulators 3 Fixing and blocking or inhibitors eej Li 4 Anti phospho protein antibody 5 HRP conjugated secondary 6 Develop with substrate or anti pan protein antibody antibody TMB Color lo do e fe A ada aS i Fig 1 Cell Based protein phosphorylation procedure 3 RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Kit Protocol lll REAGENTS AND STORAGE For up to 3 months unless otherwise stated or until expiration date STORAGE AFTER TEM COMPONENT 2 PLATE KIT INITIAL THAW A Uncoated 96 Well Microplate 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 15X Mouse Anti phospho Thr202 Tyr204 ERK1 2 G 1 1vial Concentrate 15X Mouse Anti phospho Thr180 Tyr182 p38 MAPK G 2 1vial Concentrate 63 15X Mouse Anti phospho Thr183 Tyr185 JNK DA 532e Concentrate H 1 15X Mouse Anti ERK1 2 Concentrate 1 vial H 2 15X Mouse Anti p38 MAPK Concentrate 1 vial H 3 15X Mouse Anti JNK Concentrate 1 vial 10 ul l 2 1000X HRP Conjugated Anti
8. ferent concentrations of EGF for 10 min at 37 C 0 5 Anti Phospho JNK Tyr183 Tyr185 0 4 4 Anti JNK 0 3 450 nm N 0 2 OD 0 1 0 0 EGF L 0 0 2 1 ug ml concentrations Fig 3 2 Hela cells were stimulated by different concentrations of anisomycin for 1 hour at 37 C 11 RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Kit Protocol Anti Phospho p38 MAPK Thr180 Tyr182 Anti p38 0 6 0 5 4 0 4 5 450 nm 0 3 0 2 4 OD 0 1 0 0 Anisomycin 0 0 2 1 ug ml concentrations Fig 3 3 Hela cells were stimulated by different concentrations of anisomycin for 1 hour at 37 C hEGF 0 10 30 0 10 30 Min Anti ERK1 2 Anti Phospho ERK 1 2 Thr202 Tyr204 Fig 4 1 Western blot analysis of extracts from 100 ng ml hEGF treated A431 cells Phospho ERK1 2 Thr202 Tyr204 and ERK1 2 antibodies were used in both detection assays 12 RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Kit Protocol Anisomycin 0 15 0 15 60 min Anti JNK Anti Phospho JNK Thr183 Tyr185 Fig 4 2 Western blot analysis of extracts from 1 ug ml Anisomycin treated Hela cells Anti Phospho JNK Thr183 Tyr185 and Anti JNK antibodies were used in both detection assays Anisomycin 0 15 60 0 15 60 Min Anti p38
9. nd 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 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 8 Add 200 ul of prepared 1X Quenching Buffer ITEM E into each well and incubate 20 minutes at room temperature 7 RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Kit Protocol 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
10. r 15 RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Kit Protocol NOTES 16 RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Kit Protocol This product is for research use only 2004 RayBiotech Inc 17 RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Kit Protocol
11. tion No Preparation N A E 30X Quenching Buffer Dilute 30 fold with 1X Wash 1mlofconcentrate 29 mlofwash buffer Concentrate Buffer A 30mlof 1X working solution Dilute 5 fold with distilled or 20 mlof concentrate 80 ml of water a Seld RE BUNGE Concent r l deionized water 100mlof 1X working solution 15X Mouse Anti phospho G 1 Thr202 Tyr204 ERK1 2 Concentrate A 15X Mouse Anti phospho Q G 2 Thr180 Tyr182 p38 MAPK 1 Reconstitute by adding 100 ul of 1X Concentrate Blocking Buffer to each vial 100 ul of reconstituted stock 1400 ul of lt G 3 15X Mouse Anti phospho 2 Pipette up and downto mix 1X Blocking Buffer 1 5 ml of 1X working Thr183 Tyr185 JNK Concentrate 3 Dilute reconstituted stock 15 fold with solution 2 ez 15X Mouse Anti ERK1 2 1XBlocking Buffer Concentrate H 2 15X Mouse Anti p38 MAPK Concentrate H 3 15X Mouse Anti JNK Concentrate gt Q O z of of 10 trate 9990 1X lt 1000X HRP Conjugated Anti Dilute 1000 fold with 1X Blocking aca RO i gt l 2 Blocking Buffer 10 ml of 1X working Pe Mouse IgG Concentrate Buffer solution 2 O Oo lu PQ J TMB Substrate No preparation N A K Stop Solution 5 RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Kit Protocol 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 E
12. ubate 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 l 8 Add 100 ul TMB Substrate and incubate 30 minutes at room temperature J 9 Add 50 ul Stop Solution to each well Read at 450 nm immediately 9 RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Kit Protocol VIII QUALITY CONTROL DATA Representative results 1 Seeded 100 ul of 30 000 cells A431 cells were used for measuring ERK1 2 and Hela cells were used for measuring JNK and P38 Mark into each appropriate well in microplate Cells were incubated at 37 C in 5 CO over night 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 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 10 RayBio Cell Based Human Mouse Rat ERK1 2 JNK p38 MAPK ELISA Kit Protocol 1 0 0 8 Anti Phospho ERK 1 2 Thr202 Tyr204 Wii Anti ERK1 2 0 6 450 nm 0 4 OD 0 2 WW 0 0 EGF concentrations 0 20 100 ng ml Fig 3 1 A431 cells were stimulated by dif
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