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1. Cell Based Human Mouse Rat Cell Based JNK Thr183 Tyr185 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 JNK Thr183 Tyr185 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 JNK 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 JNK Thr183 Tyr185 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 JNK Thr183 Tyr185 or Anti JNK 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 JNK Thr183 Tyr185 ELISA Kit Protocol Il HOW IT WORKS 1 Add cells 2 Treatment with stimulators 32. Fixing and blocking or inhibitors Lee LL 4 Anti phospho protein antibody 5 HRP conjugated secondary 6 Develop with substrate or anti pan protein antibody antibody TMB Color Je Jev AJ ssl _A Fig 1 Cell Based protein phosphorylation procedure 3 RayBio Human Mouse Rat Cell Based JNK Thr183 Tyr185 ELISA Kit Protocol Ill REAGENTS AND STORAGE Store entire kit at lt 20 C immediately upon arrival Kit must be used within 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 thr1g3 Tyr185 NK anie Iva p AVAS Mie H 1000X Mouse Anti JNK Concentrate 1vial 7 ul 2 vials 7 ul ea 20 C 1000X HRP Conjugated F2 Anti Mouse n eno ya TWO N 2 vials 10 pea J TMB Substrate 1 vial 12 ml 2 vials 12 ml ea ee K Stop Solution 1 vial 14 ml the 6 month expiration date Avoid repeated freeze thaw cycles For up to 3 months unless otherwise stated or until expiration date Contains 0 2 M Sulfuric Acid IV ADDITIONAL MATERIALS REQUIRED 1 Amodel cell line prote
3. H 1000X Mouse Anti JNK BR Concentrate i Dilute 1000 fold with 10 ul of concentrate 9990 ul of LX Blocking gt gt 1X Blocking Buffer buffer 10 ml of 1X working solution ao 1000X HRP Conjugated zt L2 9 E Anti Mouse IgG Concentrate ul J TMB Substrate i No Preparation N A K Stop Solution 5 RayBio Human Mouse Rat Cell Based JNK Thr183 Tyr185 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 Anisomycin hea 0021 0021 0021 0021 0 min va OO OEC KOK O OO FORO RO NOS omn OOO l le NOOO Oe OOO Me ELO OPEM wmn OOO OO OO Gi Or COG KORO OTO TOQ OO OVO GO om MODA OO EO SEO E E E Anti Phospho JNK Anti JNK Inhibitor Anti Inhibitor Thr183 Tyr185 Phospho JNK Anti JNK Thr183 Tyr185 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 6 RayBio Human Mouse Rat Cell Based JNK Thr183 Tyr185 ELISA Kit Protocol 2 Seed 100 ul of 30 000 cells into each
4. 0 000 cells into each well and incubate overnight n 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 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 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 Human Mouse Rat Cell Based JNK Thr183 Tyr185 ELISA Kit Protocol VIII QUALITY CONTROL DATA Representative results of Cell Based JNK Thr183 Tyr185 are shown below 1 Seeded 30 000 Hela cells into appropriate well in microplate Cells were incubated at 37 C in 596 CO over night 2 Added 50 ul different concentrations of anisomycin 0 0 2 or 1 ug 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 immediately Then flipped the microplate upside down and gently tapped to r
5. 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 JNK Thr183 Tyr185 ELISA Kit Protocol 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 L l of the prepared 1X HRP Conjugated secondary antibody ITEM 1 2 into each well and incubate for 1 hour at room temperature 15 Wash 4 times with 1X Wash Buffer B 16 Add 100 L l 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 JNK Thr183 Tyr185 ELISA Kit Protocol VII ASSAY PROCEDURE SUMMARY 1 Seed 3
6. RayBio Cell Based Human Mouse Rat JNK Thr183 Tyr185 Phosphorylation ELISA Kit For the semi quantitative detection of phosphorylated human mouse or rat JNK Thr183 Tyr185 and total JNK in adherent whole cell lines User Manual Revised July 16 2015 Cat CBEL JNK 1 1 plate kit Cat CBEL JNK 2 2 plate kit Cat CBEL JNK 5 5 plate kit Please read manual carefully before starting experiment Ris RayBiotech Inc the protein array pioneer comprarny 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 JNK Thr183 Tyr185 Phosphorylation ELISA Kit TABLE OF CONTENTS Mua ca AMD 2 Il How It Works 3 Ill Reagents and Storage ce 4 IV Additional Reagents Required 4 V Reagent Preparation nnnm 5 UNE CC doc tS 6 VII Assay Procedure Sumimaly esaet 9 VIII Quality Control Data nnn 10 PE Ig 12 X Troubleshooting Guide s 13 1 RayBio Human Mouse Rat Cell Based JNK Thr183 Tyr185 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 phosphatases have been extensively investigated and have been shown to be involved in signal transduction pathways The RayBio
7. emove all of excess wash buffer The protocol was continued as stated 0 5 0 5 EZ2 Anti Phospho JNK Tyr183 Tyr185 EZ Anti Phospho JNK Tyr183 Tyr185 0 4 4 Anti JNK 0 4 Anti JNK E os E os 0 2 A 0 2 e o 0 1 0 1 0 0 0 0 EGF 0 02 1 ml EGF 02 1 ug ml concentrations pg concentrations Fig 3A Hela cells were stimulated by different concentrations Fig 3B Hela cells were stimulated by different concentrations of anisomycin for 15 minutes at 37 C of anisomycin for 1 hour at 37 C 10 RayBio Human Mouse Rat Cell Based JNK Thr183 Tyr185 ELISA Kit Protocol Anisomycin 0 15 60 Anti JNK Anti Phospho JNK Thr183 Tyr185 Fig 4 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 11 RayBio Human Mouse Rat Cell Based JNK Thr183 Tyr185 ELISA Kit Protocol De IX REFERENCES Hinton D R et al 1988 J Comp Neurol 267 398 408 Fleming Y et al 2000 Biochem J 352 145 154 Michael J Clemens and Michael C 1997 Protein Phosphorylation in Cell Growth Regulation 1 Edition Mohit A et al 1995 Neuron 14 76 78 12 RayBio Human Mouse Rat Cell Based JNK Thr183 Tyr185 ELISA Kit Protocol X TROUBLESHOOTING GUIDE Problem Cause S
8. in 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 Sero Son V ae ups 4 RayBio Human Mouse Rat Cell Based JNK Thr183 Tyr185 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 ITEM COMPONENT PREPARATION EXAMPLE A Uncoated 96 Well Microplate No Preparation N A 20X Wash Buffer A Concentrate Dilute 20 fold with 25 mlof concentrate 475 ml of 20X Wash Buffer B Concentrate distilled or deionized water water 500 ml of 1X working solution Fixing Solution No Preparation N A E 30X Quenching Buffer Dilute 30 fold with 1mlof concentrate 29 ml of wash buffer 30 Concentrate 1X Wash Buffer A mlof 1X working solution Dilute 5 fold with 20mlofconcentrate 80mlof water 100 ml BID K R BIH L F Ce e at distilled or deionized water of1Xworking solution xs gt G 1000X Mouse Anti phospho Fo Thr183 Tyr185 JNK Concentrate cz
9. olution 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 JNK Thr183 Tyr185 ELISA Kit Protocol NOTES 14 RayBio Human Mouse Rat Cell Based JNK Thr183 Tyr185 ELISA Kit Protocol Note 15 RayBio Human Mouse Rat Cell Based JNK Thr183 Tyr185 ELISA Kit Protocol Note 16 RayBio Human Mouse Rat Cell Based JNK Thr183 Tyr185 ELISA Kit Protocol This product is for research use only 2004 RayBiotech Inc 17 RayBio Human Mouse Rat Cell Based JNK Thr183 Tyr185 ELISA Kit Protocol
10. well of the Uncoated 96 Well Microplate ITEM A provided and incubate overnight at 37 C with 596 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 cell culture wells Also avoid the use of vacuum suction or too forcefully tapping the microplate when discarding any solution 6 Add
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