Manual - RayBiotech, Inc.
Contents
1. Assay Diluent should be diluted 5 fold with deionized or distilled water before use into Item K vial to prepare a Positive Control Stock Solution Dissolve the powder thoroughly by a gentle mix Add 150 ul prepared Positive Control Stock Solution from the vial of Item K into a tube with 300 ul 1x Assay Diluent to prepare P 1 See 1 Positive control of part IX TYPICAL DATA for a typical result Pipette 300 ul 1x Assay Diluent into each tube Transfer 150 ul prepared P 1 into a tube with 300 ul 1x Asaay Diluent to produce a dilution series shown below Mix each tube thoroughly before the next transfer 1x Assay Diluent serves as the background RayBio Phospho TYK2 ELISA 150ul Positive Control Stock Solution 300 pl 1x re Is Is Os Os 4 If the Wash Concentrate 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 150ul 1500 150 ul 150 ul 5 Briefly spin the biotinylated antibody Item C before use Add 100 ul of 1x Assay Diluent into the vial to prepare a biotinylated anti phosphotyrosine 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 phosphotyrosine antibody should be diluted 80x with 1x Assay Diluent and used in step 4 of Part VII Assay Procedure 6 Briefly spin t2. Reagent Preparation step 5 to each well Incubate for I hour at room temperature with shaking Discard the solution Repeat the wash as in step 3 RayBio Phospho TYK2 ELISA 6 Add 100 ul of prepared 1X HRP Streptavidin solution see Reagent Preparation step 6 to each well Incubate for 45 minutes 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 1 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 3 Add 100 ul prepared 1X biotinylated phosphotyrosine antibody to each well Incubate hour at room temperature J 4 Add 100 ul prepared 1X HRP Streptavidin solution Incubate 45 minutes at room temperature RayBio Phospho TYK2 ELISA 5 Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature J 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 then subtract the average blank optical density i Positive Control Jurkat cells
3. buffer Solubilize cells at 4 x 10 cells ml in 1x Lysis Buffer we recommend adding protease and phosphatase inhibitors to lysis 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 84 70 C Avoid repeated freeze thaw cycles Thawed lysates should be kept on ice prior to use For the initial experiment we recommend to do a serial dilution 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 4 RayBio Phospho TYK2 ELISA 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 250 ul 1x Assay Diluent Item E2
4. MB One Step Substrate Reagent Item H 12 ml of 3 3 5 5 tetramethylbenzidine TMB in buffered solution 7 Stop Solution Item I 8 ml of 0 2 M sulfuric acid 8 Cell Lysate Buffer Item J 5 ml 2x cell lysis buffer not including protease and phosphatase inhibitors 9 Positive Control JktPVOO1 2 Item K 1 vial of lyophilized powder from cell lysates HI 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 Solution Item I and 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 RayBio Phospho TYK2 ELISA 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 I ml volumes Adjustable 1 25 ml pipettes for reagent preparation 100 ml and I liter graduated cylinders Distilled or deionized water Tubes to prepare sample dilutions O N DANAA WN V SAMPLE PREPARATION Cell lysates Rinse cells with PBS making sure to remove any remaining PBS before adding the lysis
5. RayBio Phospho TY K2 Kit For Measuring Phosphorylated TY K2 phosphotyrosine protein in Human Cell Lysates User Manual Feb 6 2014 RayBio Phospho TYK2 ELISA Kit Protocol Cat PEL TY K2 Y 2 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 ponp RayBiotech Inc RayBio Phospho TYK2 ELISA Kit Protocol TABLE OF CONTENTS L 2 II Material Provided pp 2 MI Se 3 IV Additional Materials Requlired 4 V 4 VI Reagent Preparation pe 5 VIL Assay Proceduites pp 7 VIII Assay Procedure Summary 8 IX cy 671 Dev 9 X Troubleshooting 0 10 11 1 RayBio Phospho TYK2 ELISA I INTRODUCTION RayBio Phospho TYK2 ELISA Enzyme Linked Immuno sorbent Assay kit is a very rapid convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in cell lysates By determining phosphor TYK2 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
6. Western Blot analysis This Sandwich ELISA kit is an in vitro enzyme linked immunosorbent assay for the measurement of human phospho TY K2 An anti TY K2 antibody has been coated onto a 96 well plate Samples are pipetted into the wells and phosphorylated and unphosphorylated TY K2 present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti phosphotyrosine antibody is used to detect only tyrosine phosphorylated protein 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 phosphor TYK2 bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm I MATERIAL PROVIDED 1 TYK2 Microplate Item A 96 wells 12 strips x 8 wells coated with anti TY K2 RayBio Phospho TYK2 ELISA 2 Wash Buffer Concentrate 20x item B 25 ml of 20x concentrated solution 3 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 4 Biotinylated anti phosphotyrosine Item C 2 vial of biotinylated anti phosphotyrosine each vial is enough to assay half microplate 5 HRP Streptavidin Concentrate Item G I vials 200 ul vial 600x concentrated HRP conjugated streptavidin 6 T
7. he HRP Streptavidin concentrate vial Item G and pipette up and down to mix gently before use HRP Streptavidin concentrate should be diluted 600 fold with 1x Assay Diluent For example Briefly spin the vial Item G and pipette up and down to mix gently Add 20 ul of HRP Streptavidin concentrate into a tube with 12 ml 1x Assay Diluent to prepare a 600 fold diluted HRP Streptavidin solution don t store the diluted solution for next RayBio Phospho TYK2 ELISA T day use Mix well 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 recommended that all samples or Positive Control should be run at least in duplicate Add 100 ul of each sample or positive control into appropriate wells Cover well with plate holder and incubate for 2 5 hours at room temperature or over night at 4 C with shaking 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 Add 100 ul of prepared 1X biotinylated anti phosphotyrosine antibody
8. the complete list RayBiotech Inc the protein array pioneer company strives to research and develop new products to meet demands of the biomedical 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 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 12 RayBio Phospho TYK2 ELISA Antibody array Protein array Pep
9. tide array ELISA Phosphorylation assay Tissue array Assay service just simply send your samples and get 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 13 RayBio Phospho TYK2 ELISA RayBio Phospho TYK2 ELISA 14 RayBio Phospho TYK2 ELISA 15 RayBio Phospho TYK2 ELISA 16 This product is for research use only 2004 RayBiotech Inc RayBio Phospho TYK2 ELISA
10. were treated with Pervanadate at 37 C for 10 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 Assay Diluent OD 450 nm P 1 P 2 P 3 P 4 P 5 Positive control dilution series RayBio Phospho TYK2 ELISA X TROUBLESHOOTING GUIDE 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 Contaminated wash buffer for proper washing If using an automated plate washer check that all ports are unobstructed b Make fresh wash buffer 4 Positive Control Low signal Improper storage of the ELISA kit Stop solution Improper primary or secondary antibody dilution a Upon receipt the kit should be stored at 20 C Store the positive control at 70 C after reconstitution b Stop solution should be added to each well before measurement and read OD immediately c Ensure correct dilution RayBio Phospho TYK2 ELISA 11 RayBio ELISA kits Choose from over 1000 ELISA kits for human mouse rat and a variety of other species Visit www raybiotech com for
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