Manual - RayBiotech, Inc.
Contents
1. 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 before measurement and read OD immediately Ensure correct dilution RayBio Phospho P53 S15 and Total P53 ELISA Kit Protocol RayBio ELISA kits Choose from over 1 000 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 simple2. 0 C for up to 6 months avoid repeated freeze thaw cycles Reconstituted Positive Control Item K should be stored at 70 C RayBio Phospho P53 S15 and Total P53 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 O NDNA 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 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 at 70 C Avoid repeated freeze thaw cycles Thawed lysates should be kept on ice prior to use For the initial experiment we recommend a serial dilution such as 5 fold to 50 fold for your cell lysates with Assay Diluent Item E2 before use Note The fold dilution of sample used depends on the abundance of phosphoryl
3. RayBio Human Mouse Rat Phospho P53 S15 and Total P53 ELISA Kit For Measuring Phosphorylated P53 S15 and Total P53 in Human Mouse and Rat Cell Lysates User Manual Revised June 16 2014 RayBio Phospho P53 S15 and Total P53 ELISA Kit Protocol Cat PEL P53 S15 T 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 P53 S15 and Total P53 ELISA Kit Protocol TABLE OF CONTENTS L MAO CU OD pac cxiadnesaentcietidiurwetnanesacneadiat 2 II Material Provided is ssicornc ceseessoneasedcwoscais 3 MI SUOTAG Cz inc awicdise cd edosneesneivnhseadeausdieiatetada 3 IV Additional Materials Required 4 V Sample Preparation c cece eee eee eee eee 4 VI Reagent Preparation 0 65 c cciccdecasasiesesaceeen 5 VII Assay Procedure o ss 5cexdcccpauvebasescenaededeaae 7 VIII Assay Procedure Summary 00eeee eee 8 IX Typical Dadi site cnccosdcnsscesuersceeesseentesesteion 9 i Positive C ntOle irera 9 ii UV Irradiation of T47D Cell Lines 10 X Troubleshooting Guide ccce eee e eee 11 1 RayBio Phospho P53 S1 5 and Total P53 ELISA Kit Protocol I INTRODUCTION RayBio Phospho P53 S15 and Total P53 ELISA kit is a very rapid convenient and sensitive assay kit that can monitor the activation or func
4. ated 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 P53 S15 and Total P53 ELISA Kit Protocol VI REAGENT PREPARATION Bring all reagents and samples to room temperature 18 25 C before use Assay Diluent Item E2 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 400 ul 1x Assay Diluent Item E2 into Item K to prepare Positive Control P 1 solution Dissolve the powder thoroughly by a gentle mix it can be removed by centrifuge if any precipitate in the solution is found Pipette 300 ul 1x Assay Diluent into each tube Use the Positive Control P 1 solution to produce a dilution series shown below Mix each tube thoroughly before the next transfer 1x Assay Diluent serves as the background See i Positive Control of part IX TYPICAL DATA for a typical result on page 9 Positive Control Item 150ul 1501 2 150 wl 400 ul lx A SBE RayBio Phospho P53 S15 and Total P53 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 m
5. ht at 4 C with shaking 96 well microplate coated with phosphorylated and pan antibodies Anti P53 S15 Anti pan P53 4 5 6 7 8 9 10 11 12 IOmmogOW 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 detection antibody anti P53 Reagent Preparation step 5 to each wells Incubate for 1 hour at room temperature with shaking 5 Discard the solution Repeat the wash as in step 3 7 RayBio Phospho P53 S15 and Total P53 ELISA Kit Protocol 6 Add 100 ul of prepared 1x HRP conjugated Streptavidin see Reagent Preparation step 6 to corresponding wells 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 J 2 Add 100 ul sample or positive control to each well Incubate 2 5 hours at room temperature or
6. l of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer 5 Briefly spin the detection antibody Item C 1 before use Add 100 ul of 1x Assay Diluent into the vial to prepare a detection 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 anti P53 antibody concentrate should be diluted 55 fold with 1x Assay Diluent and used in step 4 of Part VII Assay Procedure 6 Briefly spin the HRP conjugated Streptavidin Item G before use HRP conjugated Streptavidin concentrate should be diluted 300 fold with 1x Assay Diluent For example Briefly spin the vial Add 10 ul of HRP conjugated Streptavidin concentrate into a tube with 3 0 mL 1x Assay Diluent pipette up and down to mix gently to prepare a 300 fold diluted HRP conjugated Streptavidin solution 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 P53 S15 and Total P53 ELISA Kit Protocol 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 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 nig
7. overnight at 4 C J 3 Add 100 ul prepared primary antibody to each well Incubate 1 0 hours at room temperature 4 Add 100 ul prepared 1X HRP Conjugated antibody solution Incubate 45minutes 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 P53 S15 and Total P53 ELISA Kit Protocol IX TYPICAL DATA ELISA data analysis Average the duplicate readings for each sample or positive i Positive Control T47D cells were exposed to 50J m of UV light followed by a 4 hours recovery period Solubilize cells at 4 x 10 cells ml in Cell Lysate 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 P53 S15 and Total P53 ELISA Kit Protocol ii UV irradiation of T47D Cell Line T47D cells were untreated or exposed to 50J m of UV light followed by a 4 hours recovery period before lysis Cell lysates were analyzed using this phosphoELISA and Western Blot A ELISA 3 E 2 D E Untreated 1 mUV O 0 phospho P53 pan P53 B Western Blot Analysis eg er UV 0 50 50 J m 0 Anti P53 S15 Anti pan P53 RayBio Phospho P53 S15 and Total P53 ELISA Kit Protocol
8. phospho P53 S15 half plate red marker on left side and anti P53 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 conjugated Streptavidin Concentrate Item G Detection Antibody P53 Item C 1 2 vials of biotinylated anti P53 1 vial is enough to assay half microplate HRP conjugated Streptavidin Item G 200 ul of 300x concentrated HRP conjugated Streptavidin 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 T47DUVS001 1 Item K 1 vial of lyophilized powder from T47D cell lysate 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 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 Item G store at 2 8 C for up to one month store at 2
9. sensitive reliable and cost effective Products include Cytokine Arrays Chemokine Arrays ELISA kits Phosphotyrosine kits Recombinant Proteins Antibodies and custom services RayBio Phospho P53 S15 and Total P53 ELISA Kit Protocol This product is for research use only 2004 RayBiotech Inc 13 RayBio Phospho P53 S15 and Total P53 ELISA Kit Protocol
10. tion of important biological pathways in human mouse and rat cell lysates By determining phosphorylated P53 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 Blotting analysis This Sandwich ELISA kit is an in vitro enzyme linked immunosorbent assay for the measurement of human mouse and rat phospho P53 and total P53 An anti P53 S15 half plate red marker on left side and anti pan P53 antibody half plate black marker on right side have been coated onto a 96 well plate Samples are pipetted into the wells and phosphorylated left side or pan right side P53 present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti P53 antibody is used to detect phosphorylated P53 left side or pan P53 right side 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 P53 S15 or pan P53 bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm RayBio Phospho P53 S15 and Total P53 ELISA Kit Protocol Il MATERIAL PROVIDED 1 P53 Microplate Item A 96 wells 12 strips x 8 wells coated with anti
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