User Manual
Contents
1. 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 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 MKK6 S207 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 Positiv2. immediately Ensure correct dilution RayBio Phospho MKK6 S207 ELISA Kit Protocol RayBio ELISA kits Choose from over 1 600 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 MKK6 S207 ELISA Kit Protocol This product is for research use only 2015 RayBiotech Inc RayBio Phospho MKK6 S207 ELISA Kit Protocol
3. as in step 3 Add 100 ul of prepared 1x HRP conjugated anti rabbit IgG against anti MKK6 S207 see Reagent Preparation step 6 to each wells Incubate for hour at room temperature with shaking Discard the solution Repeat the wash as in step 3 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 Add 50 ul of Stop Solution Item I to each well Read at 450 nm immediately RayBio Phospho MKK6 S207 ELISA Kit Protocol Vill ASSAY PROCEDURE SUMMARY E LA P Nn Prepare all reagents samples and standards as instructed Add 100 ul sample or positive control to each well Incubate 2 5 hours at room temperature or overnight at 4 C Add 100 ul prepared primary antibody to each well Incubate 1 0 hours at room temperature Add 100 ul prepared 1X HRP Conjugated antibody solution Incubate 1 hour at room temperature Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature J Add 50 ul Stop Solution to each well Read at 450 nm immediately RayBio Phospho MKK6 S207 ELISA Kit Protocol IX TYPICAL DATA ELISA data analysis Average the duplicate readings for each sample or positive i Positive Control HeLa cells were treated with UV Solubilize cells at 4 x 10 cells ml in Cell Lysate Buffer Serial dilutions of lysates
4. were analyzed in this ELISA Please see step 3 of Part VI Reagent Preparation for detail OD 450 nm P 1 Pi P P 4 PO Positive control dilution series RayBio Phospho MKK6 S207 ELISA Kit Protocol ii UV Stimulation of HeLa Cell Line HeLa cells were treated or untreated with UV Cell lysates were analyzed using this phosphoELISA and Western Blot A ELISA 2 E E e S 3 E Untreated a EUV O 0 phospho MKK6 pan MKK6 B Western Blot Analysis a es Anti MKK6 S207 Anti pan MKK6 RayBio Phospho MKK6 S207 ELISA Kit Protocol X TROUBLESHOOTING GUIDE Problem Cause Solution 1 Sample signals a Too low a Sample concentration a Increasing sample is too low concentration b Too high b Sample concentration b Reducing sample is too high concentration 2 Large CV a Inaccurate pipetting a Check pipettes 3 High background a Plate is insufficiently a Review the manual washed b Contaminated wash buffer 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 O 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
5. RayBio Human Mousel Rat Phospho MKK6 S207 ELISA Kit For Measuring Phosphorylated MKK6 S207 in Human Mouse and Rat Cell Lysates User Manual Revised March 11 2015 RayBio Phospho MKK6 S207 ELISA Kit Protocol Cat PEL MKK6 S207 D 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 MKK6 S207 ELISA Kit Protocol TABLE OF CONTENTS I tegt Ehe Eege 2 IH Material Provided occcscicescverocectarewodeseigtvescs 3 LIEGE 3 IV Additional Materials Required 4 V Sample temaratean ss seu ENR EENS EUR de 4 VI Reagent Preparation 0222 00c0ssccseanasavasseceeaes 5 VII Assay e 7 VIII Assay Procedure Summary 8 IX Typical RE 9 1 Positive CORUM EE 9 i UV Stimulation of HeLa Cell Lines 10 X Troubleshooting Code 11 RayBio Phospho MKK6 S207 ELISA Kit Protocol I INTRODUCTION RayBio Phospho MKK6 S207 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 MKK6 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 effor
6. e 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 1 Positive Control of part IX TYPICAL DATA for a typical result on page 9 Positive Control Item 15041 1501 1504 400 ul 1x A T MS ef OS A Sei RayBio Phospho MKK6 S207 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 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 rabbit anti MKK6 S207 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 anti rabbit IgG Item D 1 before use HRP conjugated anti rabbit IgG co
7. ncentrate should be diluted 500 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 1x 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 MKK6 S207 ELISA Kit Protocol VII Bring all reagents to room temperature 18 25 C before use It is ASSAY PROCEDURE recommended that all samples or Positive Control should be run at least in duplicate 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 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 Add 100 ul of prepared 1x detection antibody anti MKK6 S207 Reagent Preparation step 5 to each wells Incubate for 1 hour at room temperature with shaking Discard the solution Repeat the wash
8. t 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 MKK6 An anti pan MKK6 antibody has been coated onto a 96 well plate Samples are pipetted into the wells and MKK6 present in a sample is bound to the wells by the immobilized antibody The wells are washed and rabbit anti MKK6 S207 antibody is used to detect phosphorylated MKK6 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 MKK6 S207 bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm RayBio Phospho MKK6 S207 ELISA Kit Protocol I MATERIAL PROVIDED 1 2 MKK6 Microplate Item A 96 wells 12 strips x 8 wells coated with anti pan MKK6 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 IgG Concentrate Item D Detection Antibody MKK6 S207 Item C 1 2 vials of rabbit anti MKK6 S207 1 vial is enough to assay half microplate HRP conjugated Anti rabbit IgG Item D 1 25 ul of 500x concentrated HRP conjugated anti rabbit IgG TMB One Step Subs
9. trate 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 HELAUVS001 1 Item K 1 vial of lyophilized powder from HeLa cell lysate Il 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 and G 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 RayBio Phospho MKK6 S207 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 CONNMNB WN V SAMPLE PREPARATION
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