Please read manual carefully before starting
Contents
1. 3 Fixing and blocking I 6 Develop with substrate 2 Treatment with stimulators or inhibitors M 5 HRP conjugated secondary antibody TMB Color BE an d Fig 1 Cell Based protein phosphorylation procedure 3 RayBio Cell Based EGFR Tyr1068 ELISA Kit Protocol Ill REAGENTS AND STORAGE Store entire kit at lt 20 C immediately upon arrival Kit must be used within STORAGE AFTER TEM 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 SE NETT ETT H 1000X Rabbit Anti EGFR Concentrate 1 vial 6 ul 2 vials 6 ul ea 20 C 2000X HRP Conjugated ia Anti Rabbit aoc Ivanou 2 vials 10 ul ea 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 protein tyrosine kinase inhibitors growth factors or cytokines Microplate reader capable of measuring absorbanc2. RayBio Cell Based Human EGFR Tyr1068 Phosphorylation ELISA Kit For the semi quantitative detection of phosphorylated human EGFR Tyr1068 and total EGFR in adherent whole cell lines User Manual Revised July 16 2015 Cat CBEL EGFR1068 1 1 plate kit Cat CBEL EGFR1068 2 2 plate kit Cat CBEL EGFR1068 5 5 plate kit Please read manual carefully before starting experiment RayBiotech Inc Hil 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 W Cell Based Human EGFR Tyr1068 Phosphorylation ELISA Kit TABLE OF CONTENTS l Introduction 2 ll HOW UW OS ienr 3 I Reagents and Storage 4 IV Additional Reagents Required 4 V Reagent Preparation sk cere eee ins 5 VI Assay Procedure nn 6 VII Assay Procedure Summary 9 VIII Quality Control Data css 10 IX cci Em 12 X Troubleshooting Guide nn 13 1 RayBio Cell Based EGFR Tyr1068 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 Cell Based Human EGFR Tyr1068
3. 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 Cell Based EGFR Tyr1068 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 see Section V Reagent Preparation 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 Repeat step 11 14 Add 50 ul of 1X HRP Conjugated secondary antibody ITEM I into each well and incubate for 1 hour at room temperature 15 Wash 3 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 Cell Based EGFR Tyr1068 ELISA Kit Protocol VII ASSAY PROCEDURE SUMMARY 1 Seed 20 000 cells into each well and incubate overnight l 2 Apply various treatment
4. Phosphorylation 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 EGFR Tyr1068 phosphorylation and screening the effects of various treatments inhibitors such as siRNA or chemicals or activators in cultured human cell lines By determining EGFR 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 EGFR Tyr1068 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 EGFR Tyr1068 or Anti EGFR primary antibody is pipetted into the wells and incubated The wells are washed and HRP conjugated anti rabbit IgG secondary antibody 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 Cell Based EGFR Tyr1068 ELISA Kit Protocol Il HOW IT WORKS 1 Add cells Loo 4 Anti phospho protein antibody or anti pan protein antibody m
5. ashing 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 Cell Based EGFR Tyr1068 ELISA Kit Protocol NOTES 14 RayBio Cell Based EGFR Tyr1068 ELISA Kit Protocol NOTES 15 RayBio Cell Based EGFR Tyr1068 ELISA Kit Protocol NOTES 16 RayBio Cell Based EGFR Tyr1068 ELISA Kit Protocol This product is for research use only 2004 RayBiotech Inc 17 RayBio Cell Based EGFR Tyr1068 ELISA Kit Protocol
6. d EGFR Tyr1068 ELISA Kit Protocol Anti Phospho EGFR Tyr1068 Wi Anti EGFR 0 8 oes 0 6 E Z 04 il A C o2 0 0 EGF concentrations 0 20 100 ng ml Fig 3 A431 cells were stimulated by different concentrations of EGF for 10 min at 379C Ls 0 10 0 10 Min Anti EGFR Anti phospho EGFR Tyr1068 Fig 4 Western blot analysis of extracts from 100 ng ml hEGF treated A431 cells Phospho EGFR Tyr1068 and EGFR antibodies were used in both detection assays 11 RayBio Cell Based EGFR Tyr1068 ELISA Kit Protocol IX REFERENCES Uv JE Hubbard S R et al 1994 Nature 372 746 754 Hackel P O et al 1999 Curr Opin Cell Biol 11 184 189 Levkowitz G et al 1999 Mol Cell 4 1029 1040 Zwick E et al 1999 Trends Pharmacol Sci 20 408 412 Biscardi J S et al 1999 J Biol Chem 274 8335 8343 12 RayBio Cell Based EGFR Tyr1068 ELISA Kit Protocol X TROUBLESHOOTING GUIDE Problem Cause 1 Low signal 1 Improper storage of the ELISA kit Solution m Store the kit according 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 w
7. e 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 Cell Based EGFR Tyr1068 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 each 20 fold with distilled or 25 ml of concentrate 475 ml of water 20X Wash Buffer B Concentrate deionized water 500 ml of IX working solution D Fixing Solution No Preparation N A 30X Quenching Buffer Dilute 30 fold with 1X Wash Buffer 1mlofconcentrate 29 ml of wash buffer E A Concentrate A 30mlof 1X working solution Dilute 5 fold with distilled or 20 ml of concentrate 80 ml of water t SSPIDSRInE Due CONCERTAR deionized water 100 ml of 1X working solution EE c 1000X Rabbit Anti phospho E 9 Tyr1068 EGFR Concentrate Dilute each 1000 fold with 1X Blocking a e la C Rami E 1000X Rabbit Anti EGFR Buffer E ER BEKA cz H solution lt C
8. 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 Cell Based EGFR Tyr1068 ELISA Kit Protocol 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 EGER Tyr1068 are shown below 1 Seeded 20 000 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 min at 37 C 3 Discarded the solution and wash 3 times with 1X Wash Buffer A 200 ul each immediately Then tapped the plate upside down to remove all of excess wash buffer The protocol was then followed as stated 10 RayBio Cell Base
9. nd 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 see Section V Reagent Preparation 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
10. oncentrate gt a 10 ul of concentrate 19990 ul of 1X lt x ao j zt ANN HPR kk SAAN Dilute 2000 fold with 1X Blocking Buffer Blocking Buffer 20mlof 1Xworking OE Anti Rabbit IgG Concentrate Oz solution ul q uo J TMB Substrate No Preparation N A K Stop Solution 5 RayBio Cell Based EGFR Tyr1068 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 EGF ng ml O 20 100 O 20 100 O 20 100 O 20 100 0 min OORD O O O O e O O O O COP Gs OCHO OO Simi ho Oe OHO C CC QO QOO CX OO 10 min e ONO CHOC OE GC RO QOO OO beet OVO ONO ONO PELO Set OIG OF ONG Or YO th Ta Anti Phospho Anti EGFR Inhibitor Inhibitor Anti EGFR EGFR Anti Phospho Tyr1068 EGFR Tyr1068 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 Cell Based EGFR Tyr1068 ELISA Kit Protocol 2 Seed 100 ul of 20 000 cells into each well of the Uncoated 96 Well Microplate ITEM A provided a
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