Please read manual carefully before starting experiment
Contents
1. 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 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 Cell Based STAT6 Tyr641 ELISA Kit VII ASSAY PROCEDURE SUMMARY 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 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 Human Mouse Cell Based STAT6 Tyr641 ELISA Kit 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 STAT6 Tyr641 are shown below 1 Seeded 100 ul of 30 000 A431 cells into appropriate wells of the microplate Ce2. 00 ml of 1X working solution 10ul of concentrate 4990 Ll of 1X 500X Rabbit Anti phospho gt gz G Tyr641 STAT6 Concentrate Dilute 500 fold with 1X Blocking Buffer Blocking Buffer 5mlof 1X working g Q solution ZSE 2 ul of concentrate 9998 ul of 1X cz per H SOO Mel SE ADETAN Dilute 5000 fold with 1X Blocking Buffer Blocking Buffer 10 ml of 1X working Concentrate i solution ZEN d 1000X HRP Conjugated lt 6 Anti Rabbit IgG Concentrate 10 ul of concentrate 9990 plof 1X ce B Dilute 1000 fold with 1X Blocking Buffer Blocking Buffer 10 ml of 1X working 25 2 1000X HRP Conjugated ction a T Anti Mouse IgG Concentrate J TMB Substrate i No Preparation N A K Stop Solution NOTE Briefly centrifuge 1 000g ITEMS G H and I before opening to ensure maximum 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 0O 20 100 ws jOOOlooolooo oooO 000 000 000 000 MOO o OO 00 0 000 000 000 000 000J000 000 000 000 000 000 000 999 900 000 000 um Tr ua cy T TIN OPTIONAL Tyr641 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
3. SEW Teji Cell Based Human Mouse STAT6 Tyr641 Phosphorylation ELISA Kit For the semi quantitative detection of phosphorylated human or mouse STAT6 Tyr641 and total STAT6 in adherent whole cell lines User Manual Revised July 16 2015 Cat CBEL STAT6 1 1 plate kit Cat CBEL STAT6 2 2 plate kit Cat CBEL STAT6 5 5 plate kit Please read manual carefully before starting experiment 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 STAT6 Tyr641 Phosphorylation ELISA Kit TABLE OF CONTENTS l Introduction M BMMD nn 3D DDBEE I IE Ie IM MAE AME N NN 2 INE idc 3 Ill Reagents and Storage 4 IV Additional Reagents Required e 4 V Reagent Preparation lnn 5 VI Assay Procedure nn 6 VII Assay Procedure Summary 9 VIII Quality Control Data s4 i 544y aAAA 10 IX References lt MDMDMMMDMIDJIMMDRN A 12 X Troubleshooting Guide nn 13 1 RayBio Human Mouse Cell Based STAT6 Tyr641 ELISA Kit 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 i
4. dd cells 2 Treatment with stimulators 3 Fixing and blocking or inhibitors Lee Ll gt 4 Anti phospho protein antibody 5 HRP conjugated secondary 6 Develop with substrate or anti pan protein antibody antibody TMB Color o _ LAT i E ENS n Fig 1 Cell Based protein phosphorylation procedure 3 RayBio Human Mouse Cell Based STAT6 Tyr641 ELISA Kit 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 500X Rabbit Anti phospho Tyr641 G STAT6 Concentrate 1 vial 10 pl 2 vials 10 ul ea H 5000X Mouse Anti STAT6 Concentrate 1 vial 5 ul 2 vials 5 ul ea 1000X HRP Conjugated 20 C ET Anti Rabbit IgG Concentrate ZNEITOHI 2 vials 10 ul ea 1000X HRP Conjugated E Anti Mouse IgG Concentrate Telo pl 2 vias 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
5. 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 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 Human Mouse Cell Based STAT6 Tyr641 ELISA Kit 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 exce
6. lls were incubated at 37 C in 596 CO overnight 2 Added 50 ul of different concentrations of stimulators rhEGF concentration for A431 cells O 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 washed 3 times with 1X Wash Buffer A 200 ul each immediately Then flipped the microplate upside down and gently tapped to remove all of excess wash buffer The protocol was continued as stated Phospho Stat 6 Tyr641 Total Stat 6 3 JI J N ni 4 0 EGF 0 20 100 ng ml concentrations Fig 3 A431 cells were stimulated by different concentration of recombinant human EGF for 10 min at 37 C 10 RayBio Human Mouse Cell Based STAT6 Tyr641 ELISA Kit hEGF 10 10 min Anti Phospho Stat 6 Tyr641 Anti Stat 6 Fig 4 Western blot analysis of extracts from 100 ng ml hEGF treated A431 cells Phospho Stat 6 Tyr641 and Stat 6 antibodies were used in both detection assays 11 RayBio Human Mouse Cell Based STAT6 Tyr641 ELISA Kit IX REFERENCES 1 Quelle F W et al Mol Cell Biol 15 3336 3343 1995 2 Patel K R et al J Biol Chem 271 22175 22182 1996 12 RayBio Human Mouse Cell Based STAT6 Tyr641 ELISA Kit X TROUBLESHOOTING GUIDE Problem Cause Solution 1 Low signal 1 Improper storage of 1 Sto
7. n signal transduction pathways The RayBio Cell Based Human Mouse STAT6 Tyr641 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 STAT6 Tyr641 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 STAT6 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 STAT6 Tyr641 Phosphorylation ELISA kit cells are seeded into a 96 well tissue culture plate The cells are fixed after various treatments inhibitors or activators After blocking an anti phospho STAT6 Tyr641 or anti STAT6 antibody is pipetted into the wells and incubated The wells are washed and an 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 Cell Based STAT6 Tyr641 ELISA Kit Il HOW IT WORKS 1 A
8. re 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 Cell Based STAT6 Tyr641 ELISA Kit NOTES 14 RayBio Human Mouse Cell Based STAT6 Tyr641 ELISA Kit Note 15 RayBio Human Mouse Cell Based STAT6 Tyr641 ELISA Kit Note 16 RayBio Human Mouse Cell Based STAT6 Tyr641 ELISA Kit This product is for research use only 2004 RayBiotech Inc 17 RayBio Human Mouse Cell Based STAT6 Tyr641 ELISA Kit
9. ss 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 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 Human Mouse Cell Based STAT6 Tyr641 ELISA Kit 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 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
10. stated or until expiration date Contains 0 2 M Sulfuric Acid IV ADDITIONAL MATERIALS REQUIRED 1 Amodelcell line protein tyrosine kinase inhibitors growth factors or S SE Le ae eS 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 4 RayBio Human Mouse Cell Based STAT6 Tyr641 ELISA Kit 9 Orbital shaker or oscillating rocker V REAGENT PREPARATION recovery RayBio Human Mouse Cell Based STAT6 Tyr641 ELISA Kit 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 ITEM 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 300 ml of 1Xworkingsolution D Fixing Solution No Preparation N A 30X Quenching Buffer Dilute 30 fold with 1X Wash Buffer 1mlofconcentrate 29 ml of wash buffer E Concentrate A 30mlof 1X working solution Dilute 5 fold with distilled or 20 mlof concentrate 80 mlof water j SAIPIBEKIDEDUIHSCROnGSn dE deionized water 1
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