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1. The RayBio Cell Based STAT1 Tyr701 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 STAT1 Tyr701 phosphorylation and screening the effects of various treatments inhibitors such as siRNA or chemicals or activators in cultured human and mouse cell lines By determining STAT1 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 STAT1 Tyr701 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 STAT1 Tyr701 or Anti STAT1 primary antibody is pipetted into the wells and incubated The wells are washed and HRP conjugated anti 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 STAT1 Tyr701 ELISA Kit Protocol ll HOW IT WORKS 1 Add cells 2 Treatment with stimulators 3 Fixing and bl2. 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 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 Cell Based STAT1 Tyr701 ELISA Kit Protocol VIII QUALITY CONTROL DATA Representative results of Cell Based STAT1 Tyr701 are shown below 1 Seeded 30 000 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 20 or 30 min at 37 C 3 Discarded the solution and wash 3 times with 1X Wash Buffer A 200 ul each immediately Then flipped the plate upside do
3. 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 sO OONN SEY LET ae eS 4 RayBio Cell Based STAT1 Tyr701 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 ITE M COMPONENT PREPARATION EXAMPLE A Uncoated 96 Well Microplate No Preparation N A B 20X Wash Buffer A Concentrate Dilute 20 fold with distilled or deionized 25 ml of concentrate 475 ml of water C 20X Wash Buffer B Concentrate Water 500ml of LX working solution D Fixing Solution No Preparation N A Dilute 30 fold with 1X Wash Buffer 1mlofconcentrate 29 mlofwash buffer E 30X Quenching Buffer Concentrate A 30 mlof2Xworking aokiton Dilute 5 fold with distilled or 20mlofconcentrate 80mlofwater j PA RIS RINE B Pence n at deionized water 100mlof 1X working solution gt gt G 1000X Mouse Anti phospho amp 6 Tyr701 STAT1 Concentrate 7H of concentrate 6993 Ul o
4. 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 RayBio Cell Based STAT1 Tyr701 ELISA Kit Protocol NOTES 14 RayBio Cell Based STAT1 Tyr701 ELISA Kit Protocol Note 15 RayBio Cell Based STAT1 Tyr701 ELISA Kit Protocol Note 16 RayBio Cell Based STAT1 Tyr701 ELISA Kit Protocol This product is for research use only 2004 RayBiotech Inc 17 RayBio Cell Based STAT1 Tyr701 ELISA Kit Protocol
5. well of the Uncoated 96 Well Microplate ITEM A provided and incubate overnight at 37 C with 5 CO2 6 RayBio Cell Based STAT1 Tyr701 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 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 forcef
6. RayBio Cell Based Human Mouse STAT1 Tyr701 Phosphorylation ELISA Kit For the semi quantitative detection of phosphorylated human or mouse STAT1 Tyr701 and total STAT1 in adherent whole cell lines User Manual Revised July 16 2015 Cat CBEL STAT1 1 1 plate kit Cat CBEL STAT1 2 2 plate kit Cat CBEL STAT1 5 5 plate kit Please read manual carefully before starting experiment Ris RayBiotech Inc 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 STAT1 Tyr701 ELISA Phosphorylation Kit TABLE OF CONTENTS l PCPOCUCTION MM BI B IiD EEE MM MIMMDMDDBIMDMDMIMDBDBDMIBBIEIA 2 Ma TeV CN OS TY Dm 3 I Reagents and Storage 4 IV Additional Reagents Required 4 V Reagent Preparation lnn 5 VI Assay Procedure nn 6 VII Assay Procedure SUmImary n 9 Vill Quality Control Data ass 10 IX References nnn 12 X Troubleshooting Guide nn 13 1 RayBio Cell Based STAT1 Tyr701 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
7. f 1X ee Blocking buffer 7 ml of 1X worki E 1000X Mouse Anti STAT1 age ee ee H solution lt Concentrate x Dilute 1000 fold with 1X Blocking Buffer x 2 lt A 3 10j of concentrate 9990 ul of 1X Q 9 z 9 2 7O99 HPP Conjugated Blocking buffer 10 ml of 1X working o Anti Mouse lgG Concentrate Oz solution n lt x J TMB Substrate i No Preparation N A K Stop Solution 5 RayBio Cell Based STAT1 Tyr701 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 0 20 100 0 20100 0 20100 0 20 100 ama OOOO XJ OOO tO JOON OOOO RO an O80 KK HON YC Oy OO QO QIJ TO ONO O20 KO ON 20 min SOT OOO OO POD WO OO bee WOON OO OO RC E OR 41O1620 11661041016 e OPTIONAL Anti Phospho Anti STAT1 Inhibitor Anti Inhibitor STAT1 Tyr701 Phospho STAT1 Anti STAT1 Tyr701 Fig 2 Example of plate layout for RayBio cell based assay 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 CelIBIND microplate or other poly lysine treated tissue culture plate may be used in place of Item A 2 Seed 100 ul of 30 000 cells into each
8. ocking or inhibitors oo LL __ 4 Anti phospho protein antibody 5 HRP conjugated secondary 6 Develop with substrate or anti pan protein antibody antibody TMB Color de le k Je EME A A A A A A Fig 1 Cell Based protein phosphorylation procedure 3 RayBio Cell Based STAT1 Tyr701 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 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 G a Ha lak Poety 702 1vial 7 u 2 vials 7 j l ea H 1000X Mouse Anti STAT1 Concentrate 1 vial 7 ul 2 vials 7 ul ea 20 C 1000X HRP Conjugated i F2 Anti Mouse n eyo an avalon 2vials 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 protein tyrosine kinase inhibitors
9. ully 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 the prepared 1X Quenching Buffer ITEM E into each well and incubate 20 minutes at room temperature 7 RayBio Cell Based STAT1 Tyr701 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 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 1X HRP Conjugated secondary antibody ITEM l 2 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 immediately 8 RayBio Cell Based STAT1 Tyr701 ELISA Kit Protocol VII ASSAY PROCEDURE SUMMARY
10. wn and tapped to remove all of excess wash buffer The protocol was then followed as stated EZ Anti Phospho Stat1 Tyr701 EZ Anti Phospho Stat1 Tyr701 Anti Stat1 Anti Stat1 0 6 0 6 0 5 0 5 0 4 J E 0 4 g gt Z 03 4 M a 0 3 i J S 02 0 2 0 1 0 1 0 0 0 0 EGF 0 20 100 ng ml EGF 0 20 100 ng ml concentrations concentrations Fig 3A A431 cells were stimulated by different Fig 3B A431 cells were stimulated by different concentrations of EGF for 10 minutes concentrations of EGF for 30 minutes at 37 C at 37 C 10 RayBio Cell Based STAT1 Tyr701 ELISA Kit Protocol hEGF 0 10 30 0 10 30 Min Anti stat1 Anti phospho stat1 Tyr701 Fig 4 Western blot analysis of extracts from 100 ng ml hEGF treated A431 cells Phospho stat1 Tyr701 and stat1 antibodies were used in both detection assays 11 RayBio Cell Based STAT1 Tyr701 ELISA Kit Protocol IX REFERENCES 1 Michael J Clemens and Michael C 1997 Protein Phosphorylation in Cell Growth Regulation 1 Edition 2 Fu X Y et al 1993 Cell 74 1135 3 Darnell J E 1997 Science 277 1630 12 RayBio Cell Based STAT1 Tyr701 ELISA Kit Protocol X TROUBLESHOOTING GUIDE 13 Problem Cause Solution 1 Low signal 1 Improper storage of 1 Store the kit according the ELISA
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