Manual - RayBiotech, Inc.
Contents
1. testing such as 5 fold and 100 fold dilution for your cell lysates with Assay Diluent Item E2 before use Note The fold dilution of sample used depends on the abundance of phosphorylated proteins and should be determined empiricallys More of the sample can be used if signals are too weak If signals are too strong the sample can be diluted further Cell Lysate Buffer should be diluted 2 fold with deionized or distilled water before use recommend to add protease and phosphatase inhibitors VI REAGENT PREPARATION 1 Bring all reagents and samples to room temperature 18 25 C before use 2 Item E2 Assay Diluent should be diluted 5 fold with deionized or distilled water before use 3 Preparation of Positive Control Briefly spin the Positive Control vial of Item K Add 700 ul 1x Assay Diluent Item E2 Assay Diluent should be diluted 5 fold with deionized or distilled water before use into Item K vial to prepare a Positive Control P 1 5 RayBio Phospho Stat1 Ser727 and pan Stat ELISA Kit Protocol Dissolve the powder thoroughly by a gentle mix it can be removed by centrifuge if any precipitate in the solution is found Pipette 260 ul 1x Assay Diluent into each tube Use the P 1 to produce a dilution series shown below Mix each tube thoroughly before the next transfer 1x Assay Diluent serves as the background 700 ul 1x Assay Diluent 1 Item K vial 30u 130 130 pi EEEEE 4 If the Wash Concent2. assay kit that can monitor the activation or function of important biological pathways in cell lysates By determining phosphorylated Statl protein in your experimental model system you can verify pathway activation in your cell lysates You can simultaneously measure numerous different cell lysates without spending excess time and effort in performing a Western Blot analysis This Sandwich ELISA kit is an in vitro enzyme linked immunosorbent assay for the measurement of human phospho Stat I Ser727 and pan Statl help normalize the results of phospho Stat 1 from different cell lysate being compared An anti Statl Ser727 half plate red marker on left side and anti pan Stat antibody half plate black marker on right side has been coated onto a 96 well plate Samples are pipetted into the wells and phosphorylated left side and pan right side Stat present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated Statl is used to detect phosphorylated or pan Statl After washing away unbound antibody HRP conjugated Streptavidin is pipetted to the wells The wells are again washed a TMB substrate solution is added to the wells and color develops in proportion to the amount of Stat Ser727 or pan Stat bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm 2 RayBio amp Phospho Stat1 Ser727 and pan Stat ELISA Kit Protocol
3. Cell Lysate Buffer Item J should be stored at 4 C to avoid repeated freeze thaw cycles Return unused wells to the pouch containing desiccant pack reseal along entire edge and store at 20 C Reconstituted Positive Control Item K should be stored at 70 C IV ADDITIONAL MATERIALS REQUIRED Microplate reader capable of measuring absorbance at 450 nm Protease and Phosphatase inhibitors Shaker 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 Distilled or deionized water Tubes to prepare sample dilutions DHAYNNAB WN Re V SAMPLE PREPARATION Cell lysates Rinse cells with PBS making sure to remove any remaining PBS before adding the Cell Lysate Buffer Solubilize cells at 4 x 10 cells ml in 1x Cell Lysate Buffer we recommend adding protease and phosphatase inhibitors to Cell Lysate Buffer prior to sample preparation Pipette up and down to resuspend and incubate the lysates with shaking at 2 8 C for 30 minutes Microcentrifuge at 13 000 rpm for 10 minutes at 2 8 C and transfer the supernates into a clean test tube Lysates should be used immediately or aliquoted and stored at 70 C Avoid repeated 4 RayBio amp Phospho Stat1 Ser727 and pan Stat ELISA Kit Protocol freeze thaw cycles Thawed lysates should be kept on ice prior to use For the initial experiment we recommend to do a serial dilution
4. II MATERIAL PROVIDED 1 Stat Microplate Item A 96 wells 12 strips x 8 wells coated with anti phospho Statl Ser727 half plate red marker on left side and anti Stat antibody half plate black marker on right side Wash Buffer Concentrate 20x Item B 25 ml of 20x concentrated solution Assay Diluent Item E2 15 ml of 5x concentrated buffer For diluting cell lysate sample detection antibody Item C and HRP Streptavidin Concentrate Item G Detection Antibody Stat1 Item C 2 vial of biotinylated anti Stat each vial is enough to assay half microplate HRP Streptavidin Concentrate Item G 2 vials 200 ul vial HRP conjugated streptavidin concentrate TMB One Step Substrate Reagent Item H 12 ml of 3 3 5 5 tetramethylbenzidine TMB in buffered solution Stop Solution Item I 8 ml of 0 2 M sulfuric acid Cell Lysate Buffer Item J 5 ml 2x cell lysis buffer not including protease and phosphatase inhibitors Positive Control A431S002 1 Item K I vial of lyophilized powder from A431 cell lysate IIl STORAGE Upon receipt the kit should be stored at 20 C Please use within 6 months from the date of shipment After initial use Wash Buffer Concentrate Item B Assay Diluent Item E2 TMB One Step Substrate Reagent Item H HRP Streptavidin Item G Stop 3 RayBio amp Phospho Stat1 Ser727 and pan Stat ELISA Kit Protocol Solution Item I and
5. RayBio Phospho Stat1 Ser727 and Pan Stat1 ELISA Kit For Measuring Phospho Stat1 Ser727 and Pan Stati in Human and Mouse Cell Lysates User Manual Revised Mar 1 2012 RayBio Phospho Stat1 Ser727 and Pan Stat1 ELISA Kit Protocol Cat PEL Stat1 S727 T JER mimm gt RayBiotech Inc We Provide You With Excellent Protein Array System And Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 770 206 2393 Web www raybiotech com Email info raybiotech com RayBiotech Inc RayBio Phospho Stat1 Ser727 and Pan Stat1 ELISA Kit Protocol TABLE OF CONTENTS L TO GUCO 1 asic crore ncaa rro EFoii OP Uode x DAE ea pad 2 IL Material Provded gassdrevne 3 MI SL rer 3 IV Additional Materials Required 4 V Sample Preparation rrrrrnnannnnnvrrnnnnnnnvnnr 4 VI Reagent Preparations ccc cceciascsssiesssaseserseeadonis 5 VII Assay PIOCOOBE su Sera a mex tia e oet tir deeds 7 VIII Assay Procedure Summary 9 P Ebo ND m 10 i Positive Confol uussasavmanassusmus 10 ILS ED SUIVIE EEE 11 iii Recombinant Human EGF Stimulation of A431 Cell Limes OE 12 RATES ee sake 13 XI Troubleshooting Guide rrnnnnnnannrnvvvnnnenn 14 1 RayBio amp Phospho Stat1 Ser727 and pan Stat ELISA Kit Protocol I INTRODUCTION RayBio Phospho Statl Ser727 and Pan Statl ELISA Enzyme Linked Immunosorbent Assay kit is a very rapid convenient and sensitive
6. Science 267 1990 1994 3 Hackel P O et al 1999 Curr Opin Cell Biol 11 184 189 4 Cooper J A and Howell B 1993 Cell 73 1051 1054 13 RayBio amp Phospho Stat1 Ser727 and pan Stat ELISA Kit Protocol XI TROUBLESHOOTING GUIDE dilution Problem Cause Solution 1 Sample signals a Too low a Sample concentration is a Increasing sample too low concentration b Too high b Sample concentration is b Reducing sample too high concentration 2 Large CV a Inaccurate pipetting a Check pipettes 3 High background a Plate is insufficiently a Review the manual washed for proper washing If using an automated plate washer check that all ports are unobstructed b Contaminated wash b Make fresh wash buffer buffer 4 Positive Control a Improper storage of the a Upon receipt the kit Low signal ELISA kit should be stored at 20 C Store the positive control at 70 C after reconstitution b Stop solution b Stop solution should be added to each well before measurement and read OD immediately c Improper primary or secondary antibody c Ensure correct dilution 14 RayBio amp Phospho Stat1 Ser727 and pan Stat ELISA Kit Protocol RayBio ELISA kits Over 200 ELISA kits custom ELISA kit choose from over 300 list visit www raybiotech com for details RayBiotech Inc the protein array pioneer company strives to research and develop new products to meet demands of the bi
7. ay Diluent OD 450 nm eo OT T T T T 1 P 1 P 2 P 3 P 4 P 5 Positive control dilution series 10 RayBio amp Phospho Stat1 Ser727 and pan Stat ELISA Kit Protocol ii Recombinant Human EGF Stimulation of A431 Cell Lines A431 cells were treated or untreated with 100 ng ml recombinant human EGF for 10 min Cell lysates were analyzed using this phosphoELISA and Western Blot A ELISA 3 5 3 0 mmm Untreated A431 EGF treated A431 259 2 0 4 ODz450 nm P 1 0 0 5 4 0 0 Phospho Stat1 Ser727 Total Stat1 B Western Blot Analysis 0 10 Anti phospho Stat I Anti Stat 1 Ser727 11 RayBio amp Phospho Stat1 Ser727 and pan Stat ELISA Kit Protocol iii SENSITIVITY The A431 cells were treated with 100 ng mL recombinant human EGF for 20 minutes to induce phosphorylation of Statl Serial dilutions of lysates were analyzed in this ELISA and by Western blot Immunoblots were incubated with anti phospho Stat1 Ser 727 A ELISA 1 6 1 4 1 2 1 0 0 8 0 6 0 4 0 2 0 0 450 nm OD 50 10 2 0 4 0 8 0 ug B Western Blot Analysis 50 25 12 5 6 25 3 13 1 56 0 78 0 39 0 ug 12 RayBio amp Phospho Stat1 Ser727 and pan Stat ELISA Kit Protocol X REFERENCES 1 Wen Z et al 1995 Cell 82 241 250 2 Zhang X et al 1995
8. coated with anti pan Stat I on the right side black marker Incubate for 1 hour at room temperature with shaking 7 Discard the solution Repeat the wash as in step 3 8 Add 100 ul of TMB One Step Substrate Reagent Item H to each well Incubate for 30 minutes at room temperature in the dark with shaking 9 Add 50 ul of Stop Solution Item I to each well Read at 450 nm immediately VIII ASSAY PROCEDURE SUMMARY Prepare all reagents samples and standards as instructed 2 Add 100 ul sample or positive control to each well Incubate 2 5 hours at room temperature or over night at 4 C U 3 Add 100 ul prepared biotinylated primary antibody to each well Incubate 1 hours at room temperature i 4 Add 100 ul prepared 1X HRP Streptavidin solution Incubate 1 hour at room temperature RayBio Phospho Stat1 Ser727 and pan Stat ELISA Kit Protocol 9 5 Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature 6 Add 50 ul Stop Solution to each well Read at 450 nm immediately IX TYPICAL DATA ELISA data analysis Average the duplicate readings for each sample or positive control i Positive Control A431 cells were treated with recombinant human EGF at 37 C for 20 min Solubilize cells at 4 x 10 cells ml in lysis buffer Serial dilutions of lysates were analyzed in this ELISA Please see step 3 of Part VI Reagent Preparation for detail Ass
9. data in 1 to 2 weeks Antibody array Protein array ELISA Quantibody array Antibody production highest quality with very competitive price Monoclonal antibody Recombinant antibody Polyclonal antibody Phase display Antibody angineering Antibody conjugation Recombinant protein production Assay development Array printing Contact and non contact arrayers All kinds of substrates of your choice including glass slides membranes and plates 16 RayBio amp Phospho Stat1 Ser727 and pan Stat ELISA Kit Protocol This product is for research use only 02004 RayBiotech Inc 17 RayBio amp Phospho Stat1 Ser727 and pan Stat ELISA Kit Protocol
10. mmended that all samples or Positive Control should be run at least in duplicate 2 Add 100 ul of each sample or positive control into appropriate wells see the following 96 well microplate formate Cover well with plate holder and incubate for 2 5 hours at room temperature or over night at 4 C with shaking 96 well microplate coated with phosphorylated and pan antibodies 7 RayBio amp Phospho Stat1 Ser727 and pan Stat ELISA Kit Protocol Anti Stat Ser 727 Anti pan Stat 3 4 5 6 7 8 9 10 11 12 Ionm00WwW gt 3 Discard the solution and wash 4 times with 1x Wash Solution Wash by filling each well with Wash Buffer 300 ul using a multi channel pipette or autowasher Complete removal of liquid at each step is essential to good performance After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it against clean paper towels 4 Add 100 ul of prepared 1x biotinylated Stat1 antibody Reagent Preparation step 5 to each well Incubate for 1 hour at room temperature with shaking 5 Discard the solution Repeat the wash as in step 3 6 Add 100 ul of 40 fold diluted HRP Streptavidin solution see Reagent Preparation step 7 to each well coated with anti Stat Ser 727 on the left side red marker see Assay Procedure stepl Add 100 ul of 200 fold diluted HRP 8 RayBio amp Phospho Stat1 Ser727 and pan Stat ELISA Kit Protocol Streptavidin solution to each well
11. omedical community RayBio s patent pending technology allows detection of over 180 cytokines chemokines and other proteins in a single experiment Our format is simple sensitive reliable and cost effective Products include Cytokine Arrays Chemokine Arrays ELISA kits Phosphotyrosine kits Recombinant Proteins Antibodies and custom services Antibody Array Cytokine Antibody Array Simultaneous detection up to 200 proteins cytokine chemokine growth factor adipokine angiogenic factor protease in one experiment Phosphorylation Antibody Array e RTK antibody array e EGFR phosphorylation antibody arrays Label based antibody array Simultaneous detection more than 500 proteins in one experiment Quantibody Array Quantitative measurement of multiple protein levels Protein Array ELISA Cell Based Phosphorylation ELISA Tissue MicroArray Protein Cytokine Chemokine Adiplokine Angiogenic factor Virus bacteria and infectious disease protein hormone Enzyme other Peptide Antibody Cytokine Adipokine Angiogenic factor Signal transduction Transcription factor Receptor Adhesion molecule Virus bacteria and other infectious agents Secondary antibody Tag antibody Immunoglobulin Hormone Cell surface Protease other 15 RayBio amp Phospho Stat1 Ser727 and pan Stat ELISA Kit Protocol Antibody array Protein array Peptide array ELISA Phosphorylation assay Tissue array Assay service just simply send your samples and get
12. rate 20x Item B contains visible crystals warm to room temperature and mix gently until dissolved Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer 5 Briefly spin the biotinylated antibody Item C before use Add 100 ul of 1x Assay Diluent into the vial to prepare a biotinylated antibody concentrate Pipette up and down to mix gently the concentrate can be stored at 4 C for 5 days or at 80 C for one month The biotinylated Stat antibody should be diluted 55 fold with 1x Assay Diuent and used in step 4 of Part VII Assay Procedure 6 Briefly spin the HRP Streptavidin concentrate vial Item G and pipette up and down to mix gently before use HRP Streptavidin 6 RayBio amp Phospho Stat1 Ser727 and pan Stat ELISA Kit Protocol concentrate should be diluted 40 fold or 200 fold see step 6 in page 8 for detail with 1x Assay Diluent For example Briefly spin the vial Item G and pipette up and down to mix gently Add 150ul of HRP Streptavidin concentrate into a tube with 6 ml Ix Assay Diluent to prepare a 40 fold diluted HRP Streptavidin solution don t store the diluted solution for next day use Mix well 7 Cell Lysate Buffer should be diluted 2 fold with deionized or distilled water before use recommend to add protease and phosphatase inhibitors VII ASSAY PROCEDURE 1 Bring all reagents to room temperature 18 25 C before use It is reco
Download Pdf Manuals
Related Search