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1. 1 0 1 0 oa Anti Phospho Stat3 Tyr705 Anti Fhosphosstat3 Tyr 705 Anti Stat3 Anti Stat3 08 4 E 064 c o 06 1 t d 044 9 044 024 Q 3 02 00 3 0 0 EGF concentrations 0 20 100 ng ml EGF concentrations 0 20 100 ng ml Fig 3A A431 cells were stimulated by different Fig 3B A431 cells were stimulated by different concentration concentrations of EGF for 10 min at 37 C EGF for 30 min at 37 C hEGF 0 10 30 0 10 30 Min Anti Phospho Stat3 Anti Stat3 Tyr705 Fig 4 Western blot analysis of extracts from 100 ng ml hEGF treated A43 1cells Phospho stat3 Tyr705 and stat3 antibodies were used in both detection assays 11 RayBio Cell Based STAT3 Tyr705 ELISA Kit Protocol IX REFERENCES 1 Kanai M et al 2003 Oncogene 22 548 554 2 Michael J Clemens and Michael C 1997 Protein Phosphorylation in Cell Growth Regulation 1 Edition 3 Fu X Y et al 1993 Cell 74 1135 4 Smith P D amp Crompton M R 1998 Biochem J 331 381 12 RayBio Cell Based STAT3 Tyr705 ELISA Kit Protocol X TROUBLESHOOTING GUIDE Problem Cause Solution 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 the2. 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 STAT3 Tyr705 ELISA Kit Protocol NOTES 14 RayBio Cell Based STAT3 Tyr705 ELISA Kit Protocol This product is for research use only 2004 RayBiotech Inc 15 RayBio Cell Based STAT3 Tyr705 ELISA Kit Protocol
3. EW ej Cell Based Human Mouse Rat STAT3 Tyr705 Phosphorylation ELISA Kit For the semi quantitative detection of phosphorylated human mouse or rat STAT3 Tyr705 and total STAT3 in adherent whole cell lines User Manual Revised May 10 2013 Cat CBEL STAT3 1 1 plate kit Cat CBEL STAT3 2 2 plate kit Please read manual carefully before starting experiment 4 RayBiotech Inc Hb 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 STAT3 Tyr705 Phosphorylation ELISA Kit TABLE OF CONTENTS Vie UENO UNC ee ee 2 I TION HIE OTS UL a etc 3 Ill Reagents and Storage 4 IV Additional Reagents Required 4 V Reagent Preparation nennen 5 VI Assay Procedure nnn 6 VII Assay Procedure Summary 9 VII Quality Control Data sse te 10 IX References 12 X Troubleshooting Guide 13 1 RayBio Cell Based STAT3 Tyr705 ELISA Kit Protocol Il 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 STAT3 Tyr705 ELISA kit is a very
4. ared 1X Quenching Buffer ITEM E into each well and incubate 20 minutes at room temperature 7 RayBio Cell Based STAT3 Tyr705 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 I 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 STAT3 Tyr705 ELISA Kit Protocol Vil ASSAY PROCEDURE SUMMARY 1 Seed 30 000 cells into each well and incubate overnight i 2 Apply various treatment inhibitors or activators according to manufacturer s instructions i 3 Add 100 ul of Fixing Solution into each well and incubate for 20 minutes at room temperature l 4 Add 200
5. eader capable of measuring absorbance at 450 nm 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 4 RayBio Cell Based STAT3 Tyr705 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 before opening to ensure maximum recovery 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 mlof concentrate 475 ml of water 20X Wash Buffer B Concentrate water S00 mif TX working solution 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 ml of water SUNBIGEKIIE DUSTE can Senate deionized water 100 ml of 1X working solution 5 G 500X Mouse Anti phospho fo Tyr705 STAT3 Concentrate 10 pl of concentrate 4990 pl of 1X s Dilute 500 fold with 1X Blocking Buffer Blocking Buffer 5 ml of 1X working 2 y 500X Mouse Anti STAT3 aol fiin lt Concentrate gt e 10 pl of concentrate 9990 plof 1X lt x a
6. gated secondary 6 Develop with substrate or anti pan protein antibody antibody 1MB Color de A k je DA LA L Fig 1 Cell Based protein phosphorylation procedure B RayBio Cell Based STAT3 Tyr705 ELISA Kit Protocol Ill REAGENTS AND STORAGE Store entire kit at lt 20 C immediately upon arrival Kit must be used within the 6 month expiration date Avoid repeated freeze thaw cycles STORAGE AFTER ITEM COMPONENT 1 PLATE KIT 2 PLATE KIT INITIAL THAW A Uncoated 96 Well Microplate 1 plate 2 plates Room Temperature B 20X Wash Buffer A Concentrate 1vial 30 ml C 20X Wash Buffer B Concentrate 1vial 30 ml 58 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 H 500X Mouse Anti STAT3 Concentrate 1 vial 10 ul 2 vials 10 ul ea 20 C l ev E Und 1 vial 104l 2 vials 10 pl ea J TMB Substrate 1 vial 12 ml 2 vials 12 ml ea jc K Stop Solution 1 vial 14 ml For up to 3 months unless otherwise stated or until expiration date Contains 0 2 M Sulfuric Acid IV ADDITIONAL MATERIALS REQUIRED 1 A model cell line protein tyrosine kinase inhibitors growth factors or cytokines 37 C incubator Absorbent paper SEO SO MED pes xod Xu Distilled or deionized water Orbital shaker or oscillating rocker Microplate r
7. ion 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 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 prep
8. o j za 1209 HRP Coniugareo Dilute 1000 fold with 1X Blocking Buffer Blocking Buffer 10 ml of 1X working o Anti Mouse IgG Concentrate Oz solution ul lt f o J TMB Substrate No Preparation N A K Stop Solution 5 RayBio Cell Based STAT3 Tyr705 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 20100 0 20 100 O 20 100 mn 000 000 000 000 000 000 000 000 wmn 290 000 000 000 O00 000 000 000 000 000 000 000 000 000 000 000 999 999 990 999 Um HD IDODIDOO TT o E UY Tyr705 Fig 2 Example of how to seed cells 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 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 CO gt 6 RayBio Cell Based STAT3 Tyr705 ELISA Kit Protocol NOTE The optimal cell number used will vary on the cell line and the relative amount of protein phosphorylat
9. 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 STAT3 Tyr705 phosphorylation and screening the effects of various treatments inhibitors such as siRNA or chemicals or activators in cultured human mouse and rat cell lines By determining STAT3 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 STAT3 Tyr705 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 STAT3 Tyr705 or Anti STAT3 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 STAT3 Tyr705 ELISA Kit Protocol Il HOW IT WORKS 1 Add cells 2 Treatment with stimulators 3 Fixing and blocking or inhibitors Lee Lj __ 4 Anti phospho protein antibody 5 HRP conju
10. 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 6 Add 50 ul of prepared 1X primary antibody to each well and incubate for 2 hours at room temperature 7 Add 50 ul 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 9 RayBio Cell Based STAT3 Tyr705 ELISA Kit Protocol 9 Add 50 ul Stop Solution to each well Read at 450 nm immediately VIII QUALITY CONTROL DATA Representative results of Cell Based STAT3 Tyr705 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 down and tapped to remove all of excess wash buffer The protocol was then followed as stated 10 RayBio Cell Based STAT3 Tyr705 ELISA Kit Protocol
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