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1. 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 lil ADDITIONAL MATERIALS REQUIRED 1 Amodel cell line protein tyrosine kinase inhibitors growth factors or cytokines 09 5O See eS eS 4 RayBio Cell Based Human Mouse Rat EGFR Tyr992 ELISA Kit Protocol 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 9 Orbital shaker or oscillating rocker IV REAGENT PREPARATION 5 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 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 Water 500mlof 1Xworking solution D Fixing Solution No Preparation N A E 30X Quenching Buffer Dilute 30 fold with 1X Wash Buffer 1milof concentrate 29 mlofwash butter Concentrate 30mlo
2. on Concentrate Dilute 1000 fold with 1X Blocking 2 ulof concentrate 1998 ul of 1X Buffer for Item G4 Blocking Buffer 2 ml of 1X working solution J TMB Substrate No preparation N A K Stop Solution recovery RayBio Cell Based Human Mouse Rat EGFR Tyr992 ELISA Kit Protocol V 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 20100 0 20 100 0 min L OO dJ yC YO KU 10 min 30 min OOOO QOS OOOO OO QOVWOO OD QO OO SFO OOOO OOOO OOS OOOO OOD POO IO OOD QVOVMCO OOO OOOOOO OOOO OOO COK RORI 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 6 RayBio Cell Based Human Mouse Rat EGFR Tyr992 ELISA Kit Protocol 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 overnig
3. 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 14 RayBio Cell Based Human Mouse Rat EGFR Tyr992 ELISA Kit Protocol NOTES 15 RayBio Cell Based Human Mouse Rat EGFR Tyr992 ELISA Kit Protocol Note 16 RayBio Cell Based Human Mouse Rat EGFR Tyr992 ELISA Kit Protocol This product is for research use only 2004 RayBiotech Inc 17 RayBio Cell Based Human Mouse Rat EGFR Tyr992 ELISA Kit Protocol
4. KS 1 Add cells oo 4 Anti phospho protein antibody or anti pan protein antibody 7 ae 3 Fixing and blocking Ew 6 Develop with substrate 2 Treatment with stimulators or inhibitors n 5 HRP conjugated secondary antibod IMB Color R k Je AA A A Fig 1 Cell Based protein phosphorylation procedure 3 RayBio Cell Based Human Mouse Rat EGFR Tyr992 ELISA Kit Protocol lll REAGENTS AND STORAGE Store entire kit at lt 20 C immediately upon arrival Kit must be used within 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 G 1 20X Mouse Anti phospho Tyr845 EGFR Concentrate 1 vial G 2 20X Rabbit Anti phospho Tyr992 EGFR Concentrate 1 vial G 3 20X Rabbit Anti phospho Tyr1068 EGFR Concentrate 1 vial G 4 20X Mouse Anti phospho Activated EGFR Concentrate 1 vial 20 C H 80X Rabbit Anti EGFR Concentrate 1 vial l 1 HRP Conjugated Anti Rabbit IgG Concentrate 1 vial 10 ul l 2 HRP Conjugated Anti Mouse IgG Concentrate 1 vial 10 pl J TMB Substrate 2 vials 12 ml ea ee K Stop Solution 1 vial 14 ml
5. RayBio Cell Based Human Mouse Rat EGFR Multi site Phosphorylation ELISA Sampler Kit For the semi quantitative detection of phosphorylated human mouse or rat EGFR Tyr845 Tyr992 Tyr1068 Activated and total EGFR in adherent whole cell lines User Manual Revised July 16 2015 Cat CBEL EGFR SK 2 plate kit Please read manual carefully before starting experiment RayBiotech Inc Hip 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 EGFR Multi Site Phosphorylation ELISA Sampler Kit TABLE OF CONTENTS Introduction I Reagents and Storage ee IV Additional Reagents Required V Reagent Preparation VI Assay Procedure nn VII Assay Procedure Summary VIII Quality Control Data IX References 1 RayBio Cell Based Human Mouse Rat EGFR Tyr992 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 EGFR ELISA Sampler Kit is a very rapid convenient and sensitive assay kit which can monitor the ac
6. Tyr992 ELISA Kit Protocol VI 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 L l 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 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 9 RayBio Cell Based Human Mouse Rat EGFR Tyr992 ELISA Kit Protocol 8 Add 100 ul TMB Substrate and incubate 30 minutes at room temperature l 9 Add 50 L l Stop Solution to each well Read at 450 nm immediately VII QUALITY CONTROL DATA Representative results of Cell Based EGER Tyr992 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 1
7. X 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 EZ2 Anti Phospho EGFR Tyr845 E2 Anti Phospho EGFR Tyr992 Anti EGFR Anti EGFR 1 0 0 8 0 8 4 0 6 E os E gt J D 0 4 A 0 4 a O 9 2 0 2 0 0 0 0 EGF concentrations 0 20 100 ng ml EGF concentrations 0 20 100 ng ml Fig 3 1 A431 cells were stimulated by different Fig 3 2 A431 cells were stimulated by different concentrations of EGF for 10 min at 37 C concentrations of EGF for 10 min at 37 C 10 RayBio Cell Based Human Mouse Rat EGFR Tyr992 ELISA Kit Protocol Anti Phospho EGFR Tyr1068 Anti EGFR EZ Anti Phospho EGFR activated W Anti EGFR 1 2 0 6 1 0 E E 0 8 1 m 04 2 r 0 6 Ra il o2 5 84 0 2 4 0 0 0 0 EGF concentrations 0 20 100 ng ml EGF concentrations 0 20 100 ng ml Fig 3 3 A431 cells were stimulated by different Fig 3 4 A431 cells were stimulated by different concentrations of EGF for 10 min at 37 C concentrations of EGF for 20 min at 379 C D hEGF 0 10 0 10 Min Anti EGFR Anti phospho EGFR Tyr845 Fig 4 1 Western
8. blot analysis of extracts from 100 ng ml hEGF treated A431 cells Phospho EGFR Tyr845 and EGFR antibodies were used in both detection assays 11 RayBio Cell Based Human Mouse Rat EGFR Tyr992 ELISA Kit Protocol 0 10 Min Anti EGFR Anti phospho EGFR Tyr992 Fig 4 2 Western blot analysis of extracts from 100 ng ml hEGF treated A431 cells Phospho EGFR Tyr992 and EGFR antibodies were used in both detection assays hEGF 0 10 0 10 Min Anti EGFR Anti phospho EGFR Tyr1068 Fig 4 3 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 hae hEGF 0 10 30 0 10 30 Min Anti Phospho EGFR activated Anti EGFR Fig 4 4 Western blot analysis of extracts from 100 ng ml hEGF treated A431 cells Phospho EGFR activated and EGFR antibodies were used in both detection assays 12 RayBio Cell Based Human Mouse Rat EGFR Tyr992 ELISA Kit Protocol VIII REFERENCES 1 Michael J Clemens and Michael C 1997 Protein Phosphorylation in Cell Growth Regulation 1 Edition Fu X Y et al 1993 Cell 74 1135 Darnell J E 1997 Science 277 1630 Kanai M et al 2003 Oncogene 22 548 554 Smith P D amp Crompton M R 1998 Biochem J 331 381 mB WN 13 RayBio Cell Based Human Mouse Rat EGFR Tyr992 ELISA Kit Protocol IX TROUBLESHOOTING GUIDE Problem Cause Solution
9. f 1X working solution Dilute 5 fold with distilled or 20mlof concentrate 80mlofwater k Sp migeking BUNEr CONGENIALE deionized water 100 ml of 1X working solution ej 20X Mouse Anti phospho Tyr845 EGFR Concentrate 1 Reconstitute by adding 100 y l of 1X gt G 2 e J BA 191392 Blocking Buffer to each vial 10 ul of reconstituted stock 190 pl of 1X G3 20x Rabbit Anti phosoh 2 Pipette up and down to mix Blocking Buffer 200 ul of 1X working e Ke Kn jL E NA B M 3 Dilute reconstituted stock 20 fold with solution 5 Tyr1068 EGFR Concentrate 1X Blockine Buf ng Buffer gt G4 20X Mouse Anti phospho amp Activated EGFR Concentrate 2 1 Reconstitute by adding 100 ul of 1X a Blocking Buffer to each vial 10 ul of reconstituted stock 790 ll of 1X H 80X Rabbit Anti EGFR Concentrate 2 Pipette up and down to mix Blocking Buffer 800 ul of 1X working 3 Dilute reconstituted stock 80 fold with solution 1X Blocking Buffer Dilute 2000 fold with 1X Blocking 2 ulof concentrate 3998 ul of 1X Buffer for Item G3 and Item H Blocking Buffer 4 ml of 1X working B I 1 HRP Conjugated Anti Rabbit IgG solution Q j Concentrate Dilute 5000 fold with 1X Blocking 2 ul of concentrate 9998 ul of 1X Buffer for Item G2 Blocking Buffer 10 ml of 1X working solution E Dilute 2000 fold with 1X Blocking 2 ul of concentrate 3998 ul of 1X 2 Buffer for Item G1 Blocking Buffer 4 ml of 1X working Q 1 2 HRP Conjugated Anti Mouse IgG solution
10. ht at 37 C with 5 CO3 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 100 ul of Fixing Solution ITEM D into each well and incubate for 20 minu
11. tes at room temperature NOTE The fixing solution is used to permeabilize the cells 7 Repeat wash step 5 7 RayBio Cell Based Human Mouse Rat EGFR Tyr992 ELISA Kit Protocol 8 Add 200 ul of 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 G1 G2 G3 G4 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 l 1 or l 2 into each well and incubate for 1 hour at room temperature NOTE Item l 1 is the secondary antibody for Items G2 G3 and H primary antibody Item l 2 is the secondary antibody for Items G1 and G4 primary antibody 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 EGFR
12. tivation or function of important biological pathways in cells It can be used for measuring the relative amount of EGFR Tyr845 EGFR Tyr992 EGFR Tyr1068 and EGFR Activated phosphorylation and screening the effect of various treatment inhibitors such as siRNA or chemicals or activators in cultured human mouse and rat cell lines EGFR Tyr1068 and EGFR Activated antibodies only can detect human 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 Cell Based EGFR ELISA Sampler Kit cells are seeded into a 96 well tissue culture plate The cells are fixed after various treatment inhibitors or activators After blocking anti phospho protein specific antibody or anti pan protein specific antibody primary antibody is pipetted into the wells and incubated The wells are washed HRP conjugated anti rabbit or mouse 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 Human Mouse Rat EGFR Tyr992 ELISA Kit Protocol ll HOW IT WOR

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