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1. wells 12 strips x 8 wells coated with anti pan Mek1 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 1 and Item C 2 and secondary antibody Item D 1 Concentrate Detection Antibody Mek1 Ser217 221 Item C 1 1 vial of rabbit anti phospho Mek1 Ser217 221 1 vial is enough to assay half microplate Detection Antibody Mek1 Item C 2 1 vials of rabbit anti pan Mek1 1 vial is enough to assay half microplate HRP conjugated Anti rabbit IgG Item D 1 25 pl 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 10 Positive Control HelaT003 1 Item K 1 vial of lyophilized powder from treated Hela 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 3 RayBio Phospho Mek1 Ser217 221 and pan Mek ELISA Kit Protocol Substrate Reagent Item H Stop Solution Item I and Cell Lysate Buffer Item J sh
2. RayBio Phospho Mek1 Ser217 221 and Pan Mek1 ELISA Kit For Measuring Phospho Mek1 Ser217 221 and Pan Mek1 in Human Mouse and Rat Cell Lysates User Manual Revised May 18 2012 RayBio Phospho Mek1 Ser217 221 and Pan Mek1 ELISA Kit Protocol Cat PEL Mek S217 T sms mE NEN 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 Mek1 Ser217 221 and Pan Mek1 ELISA Kit Protocol TABLE OF CONTENTS I froduetion asmvereessrarndensavorsssi 2 IL Maternal Provided uie terre P3 ne 3 MI SE Haare aker 3 IV Additional Materials Required 4 V Sample Preparation orrrvannnnnannenenannnnner 4 VL Reagent Preparation soc ore ensiscecceauineasideciea cess 5 VII Assay Procedure cc cece eecee eee eneeeneees 7 VIII Assay Procedure Summary 8 IX Typical Da ain crete taccasncrcoersteaesceenstens meawanvcneds 9 i Positive Control eee doi RR REPERI 9 ii TPA Stimulation of Hela Cell Lines 10 X RENS srede saan e E 11 XI Troubleshooting Guide suueusssse 12 1 RayBio Phospho Mek1 Ser217 221 and pan Mek1 ELISA Kit Protocol I INTRODUCTION RayBio Phospho Mek1 Ser217 221 and Pan Mek1 ELISA Enzyme Linked Immunosorbent Assay kit is a very rapid
3. al 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 13 RayBio Phospho Mek1 Ser217 221 and pan Mek ELISA Kit Protocol 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 14 RayBio Phospho Mek1 Ser217 221 and pan Mek ELISA Kit Protocol Note 15 RayBio Phospho Mek1 Ser217 221 and pan Mek1 ELISA Kit Protocol Note 16 RayBio Phospho Mek1 Ser217 221 and pan Mek1 ELISA Kit Protocol This product is for research use only 2004 RayBiotech Inc 17 RayBio Phospho Mek1 Ser217 221 and pan Mek ELISA Kit Protocol
4. convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in cell lysates By determining phosphorylated Mekl 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 Mekl Ser217 221 and pan Mek in human mouse and rat cell lysates help normalize the results of phospho Mekl from different cell lysate being compared An pan Mek1 antibody has been coated onto a 96 well plate Samples are pipetted into the wells and Mek1 present in a sample is bound to the wells by the immobilized antibody The wells are washed and anti phospho Mekl Ser217 221 or anti pan Mek1 is used to detect phosphorylated or pan Stat3 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 Mekl Ser217 221 or pan Mekl 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 Mek1 Ser217 221 and pan Mek ELISA Kit Protocol II MATERIAL PROVIDED 1 2 oo Mek1 Microplate Item A 96
5. e use recommend to add protease and phosphatase inhibitors 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 7 RayBio Phospho Mek1 Ser217 221 and pan Mek ELISA Kit Protocol 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 rabbit anti phospho Mek I Ser217 221 antibody or 1x rabbit anti pan Mek1 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 to corresponding well Incubate for over night at 4 C 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 wi
6. ediately c Improper primary or secondary antibody c Ensure correct dilution 12 RayBio Phospho Mek1 Ser217 221 and pan Mek1 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 hormone Enzyme other Peptide Antibody Cytokine Adipokine Angiogenic factor Sign
7. n below Mix each tube thoroughly before the next transfer 1x Assay Diluent serves as the background Positive Control powder saad 500 ul 1x Assay Diluent 30u 30 pl u EEEE 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 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 70 C for one month The anti phospho Mek1 Ser217 221 or anti pan Mek1 antibody should be diluted 55 fold with 1x Assay Diuent and used in step 4 of Part VII Assay Procedure 6 RayBio Phospho Mek1 Ser217 221 and pan Mek1 ELISA Kit Protocol 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 500 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 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 befor
8. ng this phosphoELISA and Western Blot A ELISA 2 0 mmm TPA Treated Hela 1 5 1 Untreated Hela OD 450 nm 0 5 0 0 Anti Mek1 Ser217 221 Anti pan Mek1 10 RayBio Phospho Mek1 Ser217 221 and pan Mek1 ELISA Kit Protocol B Western Blot Analysis 15 0 Min Anti phospho Mek1 Ser217 221 Anti pan Mek1 X REFERENCES 1 Cowley S et al 1994 Cell 77 841 852 2 Crews C M et al 1992 Science 258 478 480 3 Alessi D R et al 1994 EMBO J 13 1610 1619 11 RayBio Phospho Mek1 Ser217 221 and pan Mek1 ELISA Kit Protocol XI TROUBLESHOOTING GUIDE dilution 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 a Plate is insufficiently a Review the manual washed for proper washing If using an automated plate washer check that all ports are unobstructed b Contaminated wash b Make fresh wash buffer buffer 4 Positive Control a Improper storage of the a Upon receipt the kit Low signal ELISA kit should be stored at 20 C Store the positive control at 70 C after reconstitution b Stop solution b Stop solution should be added to each well before measurement and read OD imm
9. ould 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 oo 10g undi 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 4 RayBio Phospho Mek1 Ser217 221 and pan Mek1 ELISA Kit Protocol 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 ke
10. pt 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 E2 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 2 Item E2 Assay Diluent 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 500 ul 1x Assay Diluent Item E2 Assay 5 RayBio Phospho Mek1 Ser217 221 and pan Mek1 ELISA Kit Protocol 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 See 1 Positive Control of part IX TYPICAL DATA for a typical result in page 9 Dissolve the powder thoroughly by a gentle mix it can be removed by centrifuge if any precipitate in the solution is found Pipette 270 ul 1x Assay Diluent into each tube Use the Positive Control 1 to produce a dilution series show
11. th 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 8 RayBio Phospho Mek1 Ser217 221 and pan Mek ELISA Kit Protocol 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 U 4 Add 100 ul prepared 1x HRP conjugated anti rabbit IgG to corresponding well Incubate over night at 4 C i 5 Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature I 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 Hela cells were treated with TPA at 37 C for 15 min Solubilize cells at 4 x 10 cells ml in Cell Lysate Buffer Serial dilutions of cell lysates were analyzed in this ELISA Please see step 3 of Part VI Reagent Preparation for detail 9 RayBio Phospho Mek1 Ser217 221 and pan Mek1 ELISA Kit Protocol Assay Diluent OD 450 nm e C5 P 1 P 2 P 3 P 4 Control Positive control dilution series ii TPA Stimulation of Hela Cell Lines Hela cells were treated or untreated with TPA for 15 min Cell lysates were analyzed usi

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