Manual - RayBiotech, Inc.
Contents
1. i Positive Control THP1 cells 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 OD 450 nm PI P 2 P 3 P 4 P 5 Positive control dilution series ii Pervanadate Stimulation of U937 Cell Line U937 cells were treated or untreated with Pervanadate for 10 min at 37 C Cell lysates were analyzed using this phosphoELISA OD 450 nm Untreated Pervanadate RayBio Phospho ROS1 ELISA Kit Protocol X TROUBLESHOOTING GUIDE Problem Cause Solution 1 Sample signals a Too low a Sample concentration Increasing sample is too low concentration b Too high b Sample concentration Reducing sample is too high concentration 2 Large CV a Inaccurate pipetting Check pipettes 3 High background a Plate is insufficiently washed b Contaminated wash buffer Review the manual for proper washing If using an automated plate washer check that all ports are unobstructed Make fresh wash buffer 4 Low positive control signal a Improper storage of the ELISA kit b Stop solution c Improper primary or secondary antibody dilution Upon receipt the kit should be stored at 20 C Store the positive control at 70 C after reconstitution Stop solution should be added to each well before2. 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 10 ul prepared Positive Control Stock Solution from the vial of Item K into a tube with 400 ul 1x Assay Diluent to prepare P 1 See i 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 10ul Positive Control i 150ul 150 1 150 1 150 jil Stock Solution 400 ul S0 50 u u 1x Assay Diluent AN Ls AN RayBio Phospho ROS1 ELISA Kit Protocol 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 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 t
3. RayBio Human Phosphotyrosine ROS1 ELISA Kit For Measuring Phosphorylated ROS1 phosphotyrosine protein in Human Cell Lysates User Manual Revised April 27 2015 RayBio Phospho ROS1 ELISA Kit Protocol Cat PEL ROS1 Y A d Jopo malajala aja 8 SS OOD RayBiotech Inc ISO 13485 amp GLP Certified 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 ROS1 ELISA Kit Protocol TABLE OF CONTENTS Bavan s ala EE 2 IL Material Proyid d eeigen 3 H EE 3 IV Additional Materials Required 4 V Sample Preparapon k 4 VI Reagent Preparagon use kn 5 VII Assay Drocedure kk kk 7 VIII Assay Procedure Summarg ee 8 IX Typical DAt EE 9 X Troubleshooting Guide EU 10 1 RayBio Phospho ROS1 ELISA Kit Protocol I INTRODUCTION RayBio Phospho ROS1I ELISA 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 phospho ROS1 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
4. 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 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 RayBio Phospho ROS1 ELISA Kit Protocol Vil 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 2 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 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 anti phosphotyrosine antib
5. human phospho ROS1 An anti ROS1 antibody has been coated onto a 96 well plate Samples are pipetted into the wells and phosphorylated and unphosphorylated ROS1 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 ROS1 bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm RayBio Phospho ROS1 ELISA Kit Protocol Il MATERIAL PROVIDED 1 2 ROS1 Microplate Item A 96 wells 12 strips x 8 wells coated with anti ROS1 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 Biotinylated anti phosphotyrosine Item C 2 vial of biotinylated anti phosphotyrosine each vial is enough to assay half microplate HRP Streptavidin Concentrate Item G 1 vials 200 ul vial 600x concentrated HRP conjugated streptavidin TMB One Step Substrate Reagent Item H 12 ml of 3 3 5 5 tetramethylbenzidine TMB in buffe
6. measurement and read OD immediately Ensure correct dilution RayBio Phospho ROS1 ELISA Kit Protocol RayBio ELISA kits Choose from over 1000 ELISA kits for human mouse rat and a variety of other species Visit www raybiotech com for 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 400 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 RayBio Phospho ROS1 ELISA Kit Protocol Notes RayBio Phospho ROS1 ELISA Kit Protocol 12 This product is for research use only P 2004 RayBiotech Inc 13 RayBio Phospho ROS1 ELISA Kit Protocol
7. ody 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 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 RayBio Phospho ROS1 ELISA Kit Protocol 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 1 hour at room temperature 4 Add 100 ul prepared 1X HRP Streptavidin solution Incubate 45 minutes at room temperature 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 RayBio Phospho ROS1 ELISA Kit Protocol IX TYPICAL DATA ELISA data analysis Average the duplicate readings for each sample or positive control then subtract the average blank optical density
8. red 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 THPPVOO1 1 Item K 1 vial of lyophilized powder from THP1 cell lysates HL 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 ROS1 ELISA Kit Protocol 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 QO J Ch Q ZS WN V SAMPLE PREPARATION Cell lysates Rinse cells with PBS making sure to remove any remaining PBS before adding the lysis buffer Solubilize cells at 4 x 10 cells ml in 1x Lysi
9. s 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 at 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 empirically 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 RayBio Phospho ROS1 ELISA Kit Protocol VI REAGENT PREPARATION 1 Bring all reagents and samples to room temperature 18 25 C before use Item E2 Assay Diluent should be diluted 5 fold with deionized or distilled water before use Preparation of Positive Control Briefly spin the Positive Control vial of Item K Add 900 ul 1x Assay Diluent Item E2 Assay Diluent should be diluted 5 fold with
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