Please read manual carefully before starting
Contents
1. 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 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 juo 4 guid ce du 4 RayBio Cell Based STATA Tyr693 ELISA Kit Protocol V REAGENT PREPARATION NOTE Thaw qll reqgents to room temperature immediqtely before use If wash buffers contqin 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 1Xworking solution Fixing Solution No Preparation N A 30X Quenching Buffer Dilute 30 fold with 1X Wash Buffer 1mlofconcentrate 29 ml of wash buffer E ji Concentrate A 30mlof 1X working solution 2 Dilute 5 fold with distilled or 20 mlof concentrate 80 ml of water i PA DIGEK TEB N r r CONTENTA deionized water 100 ml of 12. in place of Item A 2 Seed 100 ul of 30 000 cells into each well of the Uncoated 96 Well Microplate ITEM A provided and incubate overnight at 37 C with 596 COz 6 RayBio Cell Based STAT4 Tyr693 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
3. 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 ju each immediately Then flipped the plate upside down and tapped to remove all of excess wash buffer The protocol was then followed as stated Phospho Stat 4 Tyr693 Total Stat 4 3 E S 2 1 A T11 e 0 EGF concentrations 20 100 ng ml 10 RayBio Cell Based STAT4 Tyr693 ELISA Kit Protocol hEGF 0 10 0 10 min Anti phospho stat4 Anti pan stat4 Tyr693 Fig 4 Western blot analysis of extracts from 100 ng ml hEGF treated A431 cells Phospho Stat4 Tyr693 and Stat4 antibodies were used in both detection assays 11 RayBio Cell Based STAT4 Tyr693 ELISA Kit Protocol IX REFERENCES 1 Zong Z et al Proc Natl Acad Sci USA 91 4806 4810 1994 2 Yu C R et al J Immunol 157 126 137 1996 3 Kaplan M H et al Nature 382 174 177 1996 12 RayBio Cell Based STAT4 Tyr693 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 treatme
4. RayBio Cell Based Human Mouse STAT4 Tyr693 Phosphorylation ELISA Kit For the semi quantitative detection of phosphorylated human or mouse STAT4 Tyr693 and total STAT4 in adherent whole cell lines User Manual Revised July 16 2015 Cat CBEL STAT4 1 1 plate kit Cat CBEL STAT4 2 2 plate kit Cat CBEL STAT4 5 5 plate kit Please read manual carefully before starting experiment RayBiotech Inc AW 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 STAT4 Tyr693 Phosphorylation ELISA Kit TABLE OF CONTENTS l Introduction MRMB 3 35p p y p D3B gt BD3DBD B BD3B 2 INE UC OS Tc 3 I Reagents and Storage 4 IV Additional Reagents Required e 4 V Reagent Preparation nnn 5 VI Assay Procedure nn 6 VII Assay Procedure Summary 9 VIII Quality Control Data 4 i 544y aaAAA 10 IX References nnn 12 X Troubleshooting Guide 13 1 RayBio Cell Based STAT4 Tyr693 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 involv
5. X working solution i 10 ul of concentrate 4990 Ll of 1X gt gt 500X Rabbit Anti phospho F amp aO G Dilute 500 fold with 1X Blocking Buffer Blocking Buffer 5 ml of 1X workin EIS Tyr693 STAT4 Concentrate 6 h B amp Z 1000X Mouse Anti STATA Concentrate m 1000X HRP Conjugated 10 pl of concentrate 9990 pl of 1X lt 2 l 1 Anti Rabbit IgG Concentrate Dilute 1000 fold with 1X Blocking Buffer Blocking Buffer 10 ml of 1X working za solution of 2 1000X HRP Conjugated nw lt Anti Mouse IgG Concentrate J TMB Substrate No Preparation N A K Stop Solution 5 RayBio Cell Based STATA Tyr693 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 0 20 100 O 20 100 O 20 100 ws 1OOO OCOO OOO OOO 000 000 000 000 MOO o OO 00 0 000 000 000 000 000J000 000 000 000 000 000 000 999 900 000 000 w2 Tr ua cy T OPTIONAL Tyr693 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 CellBIND microplate or other poly lysine treated tissue culture plate may be used
6. 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 the prepared 1X Quenching Buffer ITEM E into each well and incubate 20 minutes at room temperature 7 RayBio Cell Based STATA Tyr693 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 L l of the prepared 1X HRP Conjugated secondary anttibody 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 Item G primary antibody Item l 2 is the secondary antibody for Item H primary antibody 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 fo
7. ed in signal transduction pathways The RayBio Cell Based STAT4 Tyr693 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 STAT4 Tyr693 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 STAT4 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 STAT4 Tyr693 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 STAT4 Tyr693 or Anti STAT4 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 Cell Based STAT4 Tyr693 ELISA Kit Protocol Il HOW IT WORKS 1 Add cells ee 4 Anti phospho protein antibody
8. nts 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 Cell Based STAT4 Tyr693 ELISA Kit Protocol NOTES 14 RayBio Cell Based STAT4 Tyr693 ELISA Kit Protocol Note 15 RayBio Cell Based STAT4 Tyr693 ELISA Kit Protocol Note 16 RayBio Cell Based STAT4 Tyr693 ELISA Kit Protocol This product is for research use only 2004 RayBiotech Inc 17 RayBio Cell Based STAT4 Tyr693 ELISA Kit Protocol
9. or anti pan protein antibody dem 3 Fixing and blocking 6 Develop with substrate 2 Treatment with stimulators or inhibitors 3 5 HRP conjugated secondary antibody Je Je Je A A A A A TMB Color Fig 1 Cell Based protein phosphorylation procedure 3 RayBio Cell Based STATA Tyr693 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 1vial 2 ml F 5XBlocking Buffer Concentrate 1vial 20 ml 2 8 C 1 month 500X Rabbit Anti phospho Tyr693 A G STATA Concentrate 1 vial 10 ul 2 vials 10 ul ea H 1000X Mouse Anti STAT4 Concentrate 1 vial 7 ul 2 vials 7 ul ea 1000X HRP Conjugated i 20 C 3 nti Rabbit IgG Concentrate Suam 2 vials 10 pea 1000X HRP Conjugated at Moie IgG Concentrate ud aka 2 vals 10 pea J TMB Substrate 1 vial 12 ml 2 vials 12 ml ea 28 C 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
10. r 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 STATA Tyr693 ELISA Kit Protocol VII ASSAY PROCEDURE SUMMARY 1 Seed 30 000 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 l 9 Add 50 L l Stop Solution to each well Read at 450 nm immediately 9 RayBio Cell Based STAT4 Tyr693 ELISA Kit Protocol VIII QUALITY CONTROL DATA Representative results of Cell Based STAT4 Tyr693 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 O 20 or 100 ng ml in
Download Pdf Manuals
Related Search