Manual - RayBiotech, Inc.
Contents
1. 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 14 RayBio Phospho ERK JNK P38a 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 simple sensitive reliable and cost effective Products include Cytokine Arrays Chemokine Arrays ELISA kits Phosphotyrosine kits Recombinant Proteins Antibodies and custom services 15 RayBio Phospho ERK JNK P38a ELISA Kit Protocol This product is for research use only 2004 RayBiotech Inc 16 RayBio Phospho ERK JNK P38a ELISA Kit Protocol2. Phospho ERK JNK P38a ELISA Kit Protocol I INTRODUCTION RayBio Phospho ERK JNK P38qa ELISA Kit is a very rapid convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in human mouse and rat cell lysates By determining phosphorylated ERK 1 2 JNK and P38qa 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 ERK 1 2 total ERK1 2 phospho JNK total JNK phospho P38a and total P38qa For each target a capture antibody has been coated onto microwells Samples are pipetted into the wells and target protein present in a sample is bound to the wells by the immobilized antibody The wells are washed and a detection antibody is used to detect the captured target protein After washing away unbound antibody an HRP conjugated secondary antibody 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 target protein bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm RayBio Phospho ERK JNK P38a ELISA Kit Protocol Il MA
3. 2 Y204 Anti pan Erk1 2 Erk2 T185 Y 187 11 RayBio Phospho ERK JNK P38a ELISA Kit Protocol iii IL 1B Stimulation of HepG2 Cell Line HepG2 cells were treated or untreated with 25ng ml IL 1B for 30 min Cell lysates were analyzed using this phosphoELISA and Western Blot A ELISA 3 E 2 1o E Untreated 1 E iL 1b O 0 phospho JNK pan JNK B Western Blot Analysis 0 30 0 30 Min Anti JNK T183 Y185 Anti pan JNK 12 RayBio Phospho ERK JNK P38a ELISA Kit Protocol iiii Anisomycin Stimulation of HeLa Cell Lines HeLa cells were treated or untreated with Anisomycin for 10 min at 37 C Cell lysates were analyzed using this phosphoELISA and Western Blot A ELISA E Untreated E Anisomycin OD 450 nm phospho P38 pan P38 B Western Blot Analysis Anisomycin 0 10 0 10 Min Anti phospho p38 alpha Anti pan p38 Thr180 Tyr182 13 RayBio Phospho ERK JNK P38a 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
4. RayBio Phospho ERK JNK P38 a ELISA Kit For measuring ERK1 2 T202 Y204 JNK T183 Y185 P38a T180 Y182 and Total ERK1 2 JNK P38qa in Human Mouse and Rat Cell Lysates User Manual Revised Aug 26 2014 RayBio Phospho ERK JNK P384a ELISA Kit Protocol Cat PEL MAPK SK aaa gene eg jajaja eos 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 ERK JNK P38a ELISA Kit Protocol TABLE OF CONTENTS LPO GUCNON soit ccesaascaressnreceseorscconwsraes 2 IL Material PrOViGe Caccace caiadateteueta vite twnracaaeads 3 HI SUOEA OS cs cut sauce iotiatssedwtacsicsdateaxmecawsecaiads 3 IV Additional Materials Required 4 V Sample Preparation c cee ecee eee eee eee 4 VI Reagent PreparaG Otis cizccsenasoveedsananeesegseanes 5 VII Assay Procedure cnssnccdscntiteceasctannctucsdereute 7 VII Assay Procedure Summary 00eeee eee 8 IX Typical Data sexs crconcae eaten hieuxawais ot cece 10 i Positive CONG i is2i5 ccicsavaneceedaedesersaes 10 u EGF Stimulation of A431 Cell Lines iii IL 1b Stimulation of HepG2 Cell lines iv Anisomycin Stimulation of HeLa Cell NCS occ opaie re darren ee tie Sawanwvbedeaesneteoxadad 13 X Troubleshooting Guide cccee eee e eee RayBio
5. TERIAL PROVIDED 1 10 11 12 13 14 1d 1 Microplate Item A 96 wells 12 strips x 8 wells coated with anti pan ERK1 2 column 1 4 JNK column 5 8 and P38a column 9 12 antibodies 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 IgG or Streptavidin Concentrate Item D G Detection Antibody Erk1 2 T202 Y 204 Item C 1 1 vial of rabbit anti phospho Erk 1 2 T202 Y204 1 vial is enough to assay 3 strips Detection Antibody Erk1 2 Item C 2 1 vial of biotinylated anti pan Erk1 2 1 vial is enough to assay 3 strips Detection Antibody JNK T183 Y185 Item C 3 1 vial of rabbit anti JNK T183 Y185 1 vial is enough to assay 3 strips Detection Antibody JNK Item C 4 1 vial of biotinylated anti JNK 1 vial is enough to assay 3 strips Detection Antibody P38a T180 Y 182 Item C 5 1 vial of rabbit anti P38a T180 Y 182 1 vial is enough to assay 3 strips Detection Antibody P38a Item C 6 1 vial of biotinylated anti P38a 1 vial is enough to assay 3 strips HRP conjugated Anti rabbit IgG Item D 1 25 ul of 500x concentrated HRP conjugated anti rabbit IgG HRP conjugated Streptavidin Item G 200 ul of 100x concentrated HRP conjugated Streptavidin TMB One Step Substrate Reagent Item H 12 ml of 3 3 5 5 tetramethylbenzid
6. gent Preparation step 5 to appropriate wells Incubate for 1 hour at room temperature with shaking RayBio Phospho ERK JNK P38a ELISA Kit Protocol 5 Discard the solution Repeat the wash as in step 3 6 Add 100 ul of prepared 1x HRP conjugated anti rabbit IgG against anti phosphorylated ERK1 2 T202 Y204 JNK T183 Y 185 and P38 T180 Y 182 antibodies or HRP conjugated Streptavidin against biotinylated anti total ERK1 2 JNK and P38 antibodies see Reagent Preparation step 6 to corresponding wells 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 RayBio Phospho ERK JNK P38a 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 overnight at 4 C 3 Add 100 ul prepared primary antibody to each well Incubate 1 0 hours at room temperature J 4 Add 100 ul prepared 1X HRP Conjugated antibody solution Incubate 1 hour 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 Soluti
7. ine 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 1 vial of lyophilized powder from A431 cell lysate RayBio Phospho ERK JNK P38a ELISA Kit Protocol 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 D store at 2 8 C for up to one month store at 20 C for up to 6 months avoid repeated freeze thaw cycles 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 DArAINNB WN RK V SAMPLE PREPARATION Cell lysates Rinse cells with PBS making sure to remove any rema
8. ining 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 RayBio Phospho ERK JNK P38a ELISA Kit Protocol 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 VI REAGENT PREPARATION 1 Bring all reagents and samples to room temperature 18 25 C before use 2 Assay Diluent Item E2 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 400 ul 1
9. on to each well Read at 450 nm immediately RayBio Phospho ERK JNK P38a ELISA Kit Protocol IX TYPICAL DATA ELISA data analysis Average the duplicate readings for each sample or positive 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 Cell Lysate Buffer Serial dilutions of lysates were analyzed using phospho ERK1 2 strips in this ELISA Please see step 3 of Part VI Reagent Preparation for detail Assay Diluent OD 450 nm 0 1 1 P 41 P 2 P 3 P 4 P 5 Positive control dilution series HepG2 cells were treated with IL 1B at 37 C for 30 min Solubilize cells at 4 x 10 cells ml in Cell Lysate Buffer Serial dilutions of lysates were analyzed using phospho JNK strips in this ELISA Assay Diluent OD 450 nm P l P 2 P 3 P 4 P 5 Positive control dilution series 10 RayBio Phospho ERK JNK P38a 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 20 min Cell lysates were analyzed using this phosphoELISA and Western Blot A ELISA 2 0 mm Untreated A431 1 5 EGF treated A431 D D 1 0 ll Q O 0 5 J E 0 0 Phospho Erk1 T202 Y204 Pan Erk1 2 Erk2 T185 Y187 B Western Blot Analysis EE _ ees hEGF 20 0 20 0 Min Anti Erk1 T20
10. uent HRP conjugated Streptavidin concentrate should be diluted 100 fold with 1x Assay Diluent For example Briefly spin the vial Add 10 ul of HRP conjugated anti rabbit IgG concentrate into a tube with 5 0 mL Ix Assay Diluent pipette up and down to mix gently to prepare a 500 fold diluted HRP conjugated anti rabbit IgG 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 ERK JNK P38a 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 night at 4 C with shaking 96 well microplate coated with pan antibodies Anti ERK Anti JNK Anti P38 4 5 6 7 8 9 10 11 12 IOTIMUOW D 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 Rea
11. x 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 10 Positive Control Item 150ul 150 2 150 pl 400 ul 1x A Dive OS OS OS 398 g RayBio Phospho ERK JNK P38a 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 detection antibody Item C 1 6 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 detection antibody concentrate should be diluted 30 fold with 1x Assay Diluent and used in step 4 of Part VII Assay Procedure 6 Briefly spin the HRP conjugated anti rabbit IgG Item D 1 or Streptavidin Item G before use HRP conjugated anti rabbit IgG concentrate should be diluted 500 fold with 1x Assay Dil
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