RayBio®Phospho Erk1(T202/Y204)/ Erk2(T185/Y187) and Pan Erk1
Contents
1. ceseee scene eee 13 1 RayBio Phospho Erk1 T202 Y 204 Erk2 T185 Y 187 and pan Erk1 2 ELISA Kit Protocol I INTRODUCTION RayBio Phospho Erk1 T202 Y204 Erk2 T185 Y187 and Pan Erk1 2 ELISA Enzyme Linked Immunosorbent 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 phosphorylated Erk1 2 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 phospho Erk1 T202 Y 204 Erk2 T185 Y187 and pan Erk1 2 in human mouse and rat cell lysates help normalize the results of phospho Erk1 2 from different cell lysate being compared An pan Erk1 2 antibody has been coated onto a 96 well plate Samples are pipetted into the wells and Erk1 2 present in a sample is bound to the wells by the immobilized antibody The wells are washed and anti phospho Erk 1 T202 Y 204 Erk2 T185 Y 187 or anti pan Erk1 2 is used to detect phosphorylated or pan Erk1 2 After washing away unbound antibody HRP conjugated anti rabbit IgG is pipetted to the wells The wells are again washed a TMB substrate solution is added to the wells and color develops in propo2. 11 RayBio Phospho Erk1 T202 Y 204 Erk2 T185 Y 187 and pan Erk1 2 ELISA Kit Protocol X REFERENCES 1 Boulton TG Cobb MH Identification of multiple extracellular signal regulated kinases ERKs with antipeptide antibodies Cell Regul 1991 2 5 357 371 2 Meng J Casey PJ Activation of Gz attenuates Rap1l mediated differentiation of PC12 cells J Biol Chem 2002 277 45 43417 43424 3 Ackerley S Grierson AJ Brownlees J et al Glutamate slows axonal transport of neurofilaments in transfected neurons J Cell Biol 2000 150 1 165 175 12 RayBio Phospho Erk1 T202 Y 204 Erk2 T185 Y 187 and pan Erk1 2 ELISA Kit Protocol XI 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 Plate is insufficiently washed Contaminated wash buffer a Review the manual 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
3. 2 ELISA Kit Protocol 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 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 50 fold dilution for your cell lysates with Assay Diluent Item E 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 5 RayBio Phospho Erk1 T202 Y204 Erk2 T185 Y 187 and pan Erk1 2 ELISA Kit Protocol 2 Item D Assay Diluent should be diluted 5 fold with deionized or distilled water before use 3 Preparation of Positive Control Briefly spin
4. Diuent and used in step 4 of Part VII Assay Procedure 6 Briefly spin the HRP conjugated anti rabbit IgG Item D 1 HRP streptavidin concentrate Item G before use Pipette up and down to mix gently HRP conjugated anti rabbit IgG concentrate should be diluted 500 fold and HRP streptavidin concentrate should be diluted 120 fold with 1x Assay Diuent For example Briefly spin the vial Item D 1 and pipette up and down to mix gently Add 10 ul of HRP conjugated anti rabbit IgG concentrate into a tube with 5 0 ml 1x AssayDiluent to prepare a 500 fold diluted HRP conjugated anti rabbit IgG solution 7 Cell Lysate Buffer should be diluted 2 fold with deionized or distilled water before use recommend to add protease and phosphatase inhibitors 7 RayBio Phospho Erk1 T202 Y204 Erk2 T185 Y 187 and pan Erk1 2 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 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 l
5. Positive Control A431S002 1 Item K 1 vial of lyophilized powder from A431 cell lysate 3 RayBio Phospho Erk1 T202 Y204 Erk2 T185 Y187 and pan Erk1 2 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 E HRP Streptavidin concentrate Item G 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 1 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 OANNB WN V SAMPLE PREPARATION Cell lysates Rinse cells with PBS making sure to remove any remaining PBS before adding the Cell Lysate Buffer Solubilize 4 RayBio Phospho Erk1 T202 Y 204 Erk2 T185 Y 187 and pan Erk1
6. RayBio Phospho Erk1 T202 Y204 Erk2 T185 Y 187 and Pan Erk1 2 ELISA Kit For Measuring Phospho Erk1 T202 Y204 Erk2 T185 Y187 and Pan Erk1 2 in Human Mouse and Rat Cell Lysates User Manual Revised May 18 2012 RayBio Phospho Erk1 T202 Y204 Erk2 T185 Y187 and Pan Erk1 2 ELISA Kit Protocol Cat PEL Erk T202 002 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 Erk1 T202 Y204 Erk2 T185 Y 187 and Pan Erk1 2 ELISA Kit Protocol TABLE OF CONTENTS I OCU CO hiss oe setren cece cccacetcensieetacsenmeraterans 2 II Material Provide iscs cise cucacies so ccacscnicavnueeisenies 3 MI Storage ennnnnnnunsensensenseensenserserrsersse 4 IV Additional Materials Required 4 V Sample PreparauOts cae osc acseenbreew nese iveadwes 4 VI Reagent Preparation 2 5 20 2cveeteasatssaccreveteaeses 5 VII Assay Procedure neers 8 VIII Assay Procedure Summary 00eee eee 9 IX Typical A ca eccsteeioenseeriveecetemnaicercieerenaaenas 10 i Positive CONDO cic iocsestacnsceticiesteteieietereass 10 ii Recombinant Human EGF Stimulation of A431 Cell Lines 02 cece cece cece eee eee nnee 11 X References cccccceccececccccccecccccesseeccescees 12 XI Troubleshooting Guide
7. ast 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 rabbit anti phospho Erk1 T202 Y 204 Erk2 T185 Y 187 antibody or 1x biotinylated anti pan Erk1 2 Reagent Preparation step 5 to appropriate wells 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 conjugated anti rabbit IgG against rabbit anti phospho Erk1 T202 Y 204 Erk2 T185 Y 187 antibody or 1X HRP streptavidin against biotinylated anti pan Erk1 2 to corresponding well Incubate for 1 hour at room temperature with shaking 8 RayBio Phospho Erk1 T202 Y204 Erk2 T185 Y 187 and pan Erk1 2 ELISA Kit Protocol 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 Vill 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 over night at 4 C J 3 Add 100 ul prepared primary antibody to appropriate well Incubate 1 0 hours at room temperature 4 Add 100 ul prepared 1x HRP conjugated anti rabbit IgG or 1X HRP strep
8. e 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 14 RayBio Phospho Erk1 T202 Y204 Erk2 T185 Y 187 and pan Erk1 2 ELISA Kit Protocol Antibody array Protein array Peptide 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 15 RayBio Phospho Erk1 T202 Y204 Erk2 T185 Y 187 and pan Erk1 2 ELISA Kit Protocol 16 RayBio Phospho Erk1 T202 Y204 Erk2 T185 Y 187 and pan Erk1 2 ELISA Kit Protocol This product is for research use only mjaa ODOC PRBE aja ajm a majaj 4 2004 RayBiotech Inc 17 RayBio Phospho Erk1 T202 Y204 Erk2 T185 Y 187 and pan Erk1 2 ELISA Kit Protocol
9. reconstitution oO Stop solution should be added to each well before measurement and read OD immediately c Ensure correct dilution 13 RayBio Phospho Erk1 T202 Y204 Erk2 T185 Y 187 and pan Erk1 2 ELISA Kit Protocol RayBio ELISA kits Over 200 ELISA kits custom ELISA kit choose from over 500 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 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 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 hormon
10. rtion to the amount of Erk 1 T202 Y 204 Erk2 T185 Y 187 or pan Erk1 2 bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm 2 RayBio Phospho Erk1 T202 Y204 Erk2 T185 Y 187 and pan Erk1 2 ELISA Kit Protocol I MATERIAL PROVIDED 1 Erk1 2 Microplate Item A 96 wells 12 strips x 8 wells coated with anti pan Erk1 2 antibody 2 Wash Buffer Concentrate 20x Item B 25 ml of 20x concentrated solution 3 Assay Diluent Item E 15 ml of 5x concentrated buffer For diluting cell lysate sample detection antibody Item C 1 and C 2 and secondary antibody Item D 1 and Item D 2 Concentrate 4 Detection Antibody Erk1 T202 Y 204 Erk2 T185 Y 187 Item C 1 1 vial of rabbit anti phospho Erk 1 T202 Y204 Erk2 T185 Y 187 1 vial is enough to assay half microplate 5 Detection Antibody Erk1 2 Item C 2 1 vial of biotinylated anti pan Erk1 2 1 vial is enough to assay half microplate 6 HRP conjugated Anti rabbit IgG Item D 1 25 ul of 500x concentrated HRP conjugated anti rabbit IgG 7 HRP Streptavidin concentrate Item G 200 ul of 100 fold HRP Streptavidin concentrate 8 TMB One Step Substrate Reagent Item H 12 ml of 3 3 5 5 tetramethylbenzidine TMB in buffered solution 9 Stop Solution Item I 8 ml of 0 2 M sulfuric acid 10 Cell Lysate Buffer Item J 5 ml 2x cell lysis buffer not including protease and phosphatase inhibitors 11
11. tavidin solution to corresponding well Incubate hour at room temperature 5 Add 100 ul TMB One Step Substrate Reagent to each well 9 RayBio Phospho Erk1 T202 Y204 Erk2 T185 Y 187 and pan Erk1 2 ELISA Kit Protocol Incubate 30 minutes at room temperature ul 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 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 in this ELISA Please see step 3 of Part VI Reagent Preparation for detail Assay Diluent OD 450 nm 0 1 T P 1 P 2 P 3 P 4 P 5 Positive control dilution series 10 RayBio Phospho Erk1 T202 Y204 Erk2 T185 Y 187 and pan Erk1 2 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 EE Untreated A431 1 5 EGF treated A431 450 nm 1 0 OD 0 5 4 0 0 Phospho Erk1 T202 Y204 Pan Erk1 2 Erk2 T185 Y187 B Western Blot Analysis oe hEGF 20 0 20 0 Min Anti Erk1 T202 Y204 Anti pan Erk1 2 Erk2 T185 Y 187
12. the Positive Control vial of Item K Add 600 ul 1x Assay Diluent Item E Assay Diluent should be diluted 5 fold with deionized or distilled water before use into Item K vial 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 400 ul 1x Assay Diluent into each tube Use the Positive Control stock solution to produce a dilution series shown below Mix each tube thoroughly before the next transfer 1x Assay Diluent serves as the background See 1 Positive Control of part IX TYPICAL DATA for a typical result in page 9 Positive Control Item K 600 ul 1x Assay Diluent 200u 200 200 ul BeBe 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 6 RayBio Phospho Erk1 T202 Y 204 Erk2 T185 Y 187 and pan Erk1 2 ELISA Kit Protocol 5 Briefly spin the detection antibody Item C 1 or Item C 2 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 anti phospho Erk1 T202 Y204 Erk2 T185 Y 187 or biotinylated anti pan Erk1 2 antibody should be diluted 55 fold with 1x Assay
Download Pdf Manuals
Related Search