RayBio®Phospho-p38 alpha MAPK (Thr180
Contents
1. m O 05 0 0 p38 alpha MAPK Thr180 Tyr182 pan p38 alpha MAPK B Western Blot Analysis Anisomycine 0 10 0 10 Min Anti phospho p38 alpha MAPK Anti pan p38 MAPK Thr180 Tyr182 11 RayBio Phospho p38 alpha MAPK Thr180 Tyr182 ELISA Kit Protocol X TROUBLESHOOTING GUIDE Problem Cause Solution 1 Sample signals a T00 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 reconstitution b Stop solution should be added to each weli before measurement and read OD immediately c Ensure correct dilution 12 RayBio Phospho p38 alpha MAPK Thr180 Tyr182 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 pioneer2. Tyr182 ELISA Kit Protocol 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 14 RayBio Phospho p38 alpha MAPK Thr180 Tyr182 ELISA Kit Protocol Note 15 RayBio Phospho p38 alpha MAPK Thr180 Tyr182 ELISA Kit Protocol Note 16 RayBio Phospho p38 alpha MAPK Thr180 Tyr182 ELISA Kit Protocol This product is for research use only 02004 RayBiotech Inc 17 RayBio Phospho p38 alpha MAPK Thr180 Tyr182 ELISA Kit Protocol
3. 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 7 RayBio Phospho p38 alpha MAPK Thr180 Tyr182 ELISA Kit Protocol 4 Add 100 ul of prepared 1X anti p38 alpha MAPK antibody Reagent Preparation step 5 to each well Incubate for I 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 solution see Reagent Preparation step 6 to each well Incubate for 2 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 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 tem
4. RayBio Phospho p38 alpha MAPK Thr180 Tyr182 ELISA Kit For Measuring Phosphorylated p38 alpha MAPK Thr180 Tyr182 in Human and Mouse Cell Lysates User Manual Revised Mar 1 2012 RayBio p38 alpha MAPK Thr180 Tyr182 ELISA Kit Protocol Cat PEL P38 T180 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 p38 alpha MAPK Thr180 Tyr182 ELISA Kit Protocol TABLE OF CONTENTS Me POG CTO soc crrncncancdnesceueneyateecunsce descend 2 II Material Provided 3 III Storage 3 IV Additional Materials Reguired 4 V Sample Preparation 4 VI Reagent Preparation 5 VII Assay Procedure 7 VIII Assay Procedure Summary 8 IK Typical Data keke keke 9 iy Positive Control Aa 9 ii Anisomycin Stimulation of Hela Cell Lines 10 X Troubleshooting Guide 12 1 RayBio Phospho p38 alpha MAPK Thr180 Tyr182 ELISA Kit Protocol I INTRODUCTION RayBio Phospho p38 alpha MAPK Thr180 Tyr182 ELISA Enzyme Linked Immunosorbent Assay kit is a very rapid convenient and sensit
5. Thr180 Tyr182 antibody 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 Anti rabbit IgG Item D 1 Detection Antibody p38 alpha MAPK Item C 2 vial of rabbit anti p38 alpha MAPK each vial is enough to assay half microplate HRP conjugated Anti rabbit IgG Item D 1 25 ul of 500x HRP conjugated anti rabbit IgG concentrate 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 HelaA003 1 Item K 2 vials of lyophilized powder from treated Hela cell lysate III 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 3 RayBio Phospho p38 alpha MAPK Thr180 Tyr182 ELISA Kit Protocol 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 a
6. 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 STAT1 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 Antibody array Protein array Peptide array ELISA Phosphorylation assay Tissue array 13 RayBio Phospho p38 alpha MAPK Thr180
7. ive assay kit that can monitor the activation or function of important biological pathways in human and mouse cell lysates By determining phosphorylated p38 alpha MAPK 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 and mouse phospho p38 alpha MAPK Thr180 Tyr182 An anti phospho p38 MAPK Thr180 Tyr182 antibody has been coated onto a 96 well plate Samples are pipetted into the wells and phosphorylated p38 MAPK Thr180 Tyr182 present in a sample is bound to the wells by the immobilized antibody The wells are washed and anti p38 alpha MAPK antibody is used to detect phosphorylated p38 alpha MAPK 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 proportion to the amount of p38 alpha MAPK Thr180 Tyr182 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 p38 alpha MAPK Thr180 Tyr182 ELISA Kit Protocol II MATERIAL PROVIDED 1 Za Microplate Item A 96 wells 12 strips x 8 wells coated with anti phospho p38 MAPK
8. perature or over night at 4 C J 8 RayBio Phospho p38 alpha MAPK Thr180 Tyr182 ELISA Kit Protocol 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 anti rabbit IgG solution Incubate 2 hour at room temperature J 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 i Positive Control Hela cells were treated with Anisomycin at 37 C for 10 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 9 RayBio Phospho p38 alpha MAPK Thr180 Tyr182 ELISA Kit Protocol Assay Diluent 10 14 Cc O LO 6 o1 0 01 T T Il Positive control dilution series ii 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 10 RayBio Phospho p38 alpha MAPK Thr180 Tyr182 ELISA Kit Protocol A ELISA 1 5 mm Untreated Hela Anisomycin treated Hela E 1 0 f O LO lt il
9. t 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 I 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 Sj N QES WN V SAMPLE PREPARATION Cell lysates Rinse cells with PBS making sure to remove any remaining PBS before adding the Cell Lysate Buffer Solubilize 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 4 RayBio Phospho p38 alpha MAPK Thr180 Tyr182 ELISA Kit Protocol 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 Ix Assay Diluent I
10. tem 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 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 Briefly spin the Positive Control vial of Item K Add 400 ul 1x Assay Diluent Item E2 Assay Diluent should be diluted 5 fold with deionized or distilled water before use into Item K vial to prepare a 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 See i Positive Control 5 RayBio Phospho p38 alpha MAPK Thr180 Tyr182 ELISA Kit Protocol of part IX TYPICAL DATA for a typical result in page 9 Pipette 240 ul 1x Assay Diluent into each tube Use the Positive Control P 1 to produce a dilution series shown below Mix each tube thoroughly before the next transfer 1x Assay Diluent serves as the background Vial of Item K 400 ul 1x Assay Diluent 1204 120 120 YS OS OS Os 2985 4 If the Wash Concentrate 20
11. x 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 p38 alpha MAPK Item C 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 The detection antibody should be diluted 55 fold with 1x Assay Diuent and used in step 4 of Part VII Assay Procedure 6 Briefly spin the HRP conjugated anti rabbit IgG Item D 1 before use Pipette up and down to mix gently HRP conjugated anti rabbit IgG concentrate should be diluted 1 000 6 RayBio Phospho p38 alpha MAPK Thr180 Tyr182 ELISA Kit Protocol I VIL fold with 1x Assay Diuent For example Briefly spin the vial ItemD and pipette up and down to mix gently Add 10 ul of HRP conjugated anti rabbit IgG concentrate into a tube with 10 ml Ix Assay Diluent to prepare a 1 000 fold diluted HRP conjugated anti rabbit IgG solution Cell Lysate Buffer should be diluted 2 folds with deionized or distilled water before use recommend to add protease and phosphatase inhibitors ASSAY PROCEDURE 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
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