Manual - RayBiotech, Inc.
Contents
1. 4 0 3 5 4 3 0 2 5 1 2 0 1 5 1 0 0 5 0 0 OD 450 nm mm Untreated A431 gt EGF treated A431 Western Blot Phospho EGFR Ser1070 EGFR 0 10 gn 0 10 Min Anti phospho EGFR Anti EGFR Ser1070 12 RayBio Phospho EGFR Ser1070 and pan EGFR ELISA Kit Protocol iii SENSITIVITY The A431 cells were treated with 100 ng mL recombinant human EGF for 20 minutes to induce phosphorylation of EGF R Serial dilutions of lysates were analyzed in this ELISA and by Western blot Immunoblots were incubated with anti phospho EGFR Ser 1070 ELISA 3 5 3 0 2 5 1 2 0 1 5 1 0 0 5 0 0 r r r i e 1 0 2 0 04 0 008 0 0016 O ug OD 450 nm Western Blot Se gt 0 X eer 50 25 12 5 6 25 3 13 1 56 0 78 0 39 0 2 O 13 RayBio Phospho EGFR Ser1070 and pan EGFR ELISA Kit Protocol X REFERENCES 1 Hackel P O et al 1999 Curr Opin Cell Biol 11 184 189 2 Alroy I and Y Yarden 1997 FEBS Lett 410 83 3 Cooper J A and Howell B 1993 Cell 73 1051 1054 4 Riedemann J et al 2007 Biochem Biophys Res Commun 355 707 14 RayBio Phospho EGFR Ser1070 and pan EGFR 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 h2. Diluent serves as the background Positive Control Item K vial 800 ul 1x Assay Diluent 150ul 150 ul 150 ul 150 ul BEBE 4 If the Wash Concentrate 20x Item B contains visible crystals warm to room temperature and mix gently until dissolved Dilute 6 RayBio Phospho EGFR Ser1070 and pan EGFR ELISA Kit Protocol 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer 5 Briefly spin the anti phospho EGFR Ser 1070 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 It can be used within one month If store at 80 C Avoid repeated freeze thaw cycles The detection antibody concentrate should further be diluted 60 folds 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 Pipette up and down to mix gently HRP conjugated anti rabbit IgG concentrate should be diluted 1 000 folds with 1x Assay Diuent 7 Briefly spin the Detection Antibody vial Item L 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 It can be used within one month If store at 80 C Avoid repeated freeze thaw cycles The detection antibody c
3. GFR ELISA Kit Protocol I MATERIAL PROVIDED 1 2 EGFR Microplate Item A 96 wells 12 strips x 8 wells coated with monoclonal anti human EGFR Wash Buffer Concentrate 20x Item B 25 ml of 20x concentrated solution Anti phospho EGFR Ser 1070 Item C Ivial rabbit anti human EGFR Ser 1070 HRP conjugated Anti rabbit IgG Item D 1 25 ul of 1000x concentrated HRP conjugated anti rabbit IgG Biotinylated Anti EGFR Item L I vial goat biotinylated anti human EGFR HRP Streptavidin concentrate Item G 200 ul of 600 fold concentrated HRP Streptavidin concentrate Assay Diluent Item E2 15 ml of 5x concentrated buffer For diluting cell lysate Biotinylated antibody Item L HRP conjugated anti rabbit IgG Item D 1 and HRP Streptavidin Item G diluent 8 TMB One Step Substrate Reagent Item H 12 ml of 9 10 11 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 Lysate Buffer not including protease and phosphatase inhibitors Positive Control A431S001 1 Item K 1 vial of lyophilized powder from A431 cell lysate 3 RayBio Phospho EGFR Ser1070 and pan EGFR 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 HRP conju
4. RayBio Phospho EGFR Ser1070 and Pan EGFR ELISA Kit For Measuring Phosph EGFR Ser 1070 and Pan EGFR in Human Cell Lysates User Manual Revised Mar 15 2012 RayBio Phospho EGFR Ser 1070 and Pan EGFR ELISA Kit Protocol Cat PEL EGFR S1070 T JER ODO gt 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 EGFR Ser 1070 and Pan EGFR ELISA Kit Protocol TABLE OF CONTENTS I INMOGUCHOM 224 ccscecersuctesievetwxsGaussusyeereareeds 2 I Material Provided ics ccswcceceseesiuwasucwedesesindearaes 3 MI Jr 4 IV Additional Materials Required 04 4 V Sample Prep ratonuonsetmudnrndene 5 VI Reagent Preparation ccc cece cence ee eens 6 VIL Assay Procedute smssssrrenanessasenenedanes kven 8 VIII Assay Procedure Summary 000eee eee 10 IX TU 10 i Positive ControlLnumrgesrinskeasidvdnvnieden 11 ii Recombinant Human EGF Stimulation of A431 Cell IMCS so sarioccacenmiaewewdandooyedianessys 11 iii S NDAG 13 X AREMENCSesickiecscctetceomsieuisbinsiersamnenn een 14 XI Troubleshooting Guide cseee cece eee 15 1 RayBio Phospho EGFR Ser1070 and pan EGFR ELISA Kit Protocol I INTRODUCTION RayBio Human Phospho EGFR Ser 1070 and Pan EGFR ELISA Enzyme Linked Imm
5. amples and standards as instructed 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 each well Incubate 1 5 hour at room temperature 4 Add 100 ul prepared secondary antibody solution Incubate 1 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 10 RayBio Phospho EGFR Ser1070 and pan EGFR ELISA Kit Protocol IX TYPICAL DATA ELISA data analysis Average the duplicate readings for each sample or positive control then subtract the average blank optical density 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 10 4 E 14 Cc O LO pi Tan 0 01 i I I I I P 1 P 2 P 3 P 4 P 5 Positive control dilution series 11 RayBio Phospho EGFR Ser1070 and pan EGFR 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 10 min Cell lysates were analyzed using this phosphoELISA and Western Blot ELISA
6. gated Anti rabbit IgG Item D 1 Assay Diluent Item E2 HRP Streptavidin 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 Reconstituted Positive Control Item K should be stored at 70 PG 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 I liter graduated cylinders Distilled or deionized water Tubes to prepare sample dilutions OrANMNB WN 4 RayBio Phospho EGFR Ser1070 and pan EGFR ELISA Kit Protocol 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
7. igh 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 immediately c Improper primary or secondary antibody c Ensure correct dilution 15 RayBio Phospho EGFR Ser1070 and pan EGFR 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 detect
8. ion 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 Signal transduction Transcription factor Receptor Adhesion molecule Virus bacteria and other infectious agents Secondary antibody Tag antibody Immunoglobulin Hormone Cell surface Protease other 16 RayBio Phospho EGFR Ser1070 and pan EGFR ELISA Kit Protocol This product is for research use only A 2004 RayBiotech Inc 17 RayBio Phospho EGFR Ser1070 and pan EGFR ELISA Kit Protocol
9. oncentrate should be diluted 200 folds with 1x Assay Diluent and used in step 4 of Part VI Assay Procedure OO Briefly spin the HRP Streptavidin concentrate vial Item G and pipette up and down to mix gently before use since precipitation may form during storage HRP Streptavidin 7 RayBio Phospho EGFR Ser1070 and pan EGFR ELISA Kit Protocol VIL Go concentrate should be diluted 600 fold with 1x Assay Diluent For example Briefly spin the vial Item G and pipette up and down to mix gently Add 20 ul of HRP Streptavidin concentrate into a tube with 12 ml 1x Assay Diluent B to prepare a 600 fold diluted HRP Streptavidin solution don t store the diluted solution for next day use Mix well 9 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 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 perf
10. ormance After the last wash remove any remaining Wash Buffer by aspirating or 8 RayBio Phospho EGFR Ser1070 and pan EGFR ELISA Kit Protocol decanting Invert the plate and blot it against clean paper towels 4 Add 100 ul of 1x anti phospho EGFR Ser 1070 to corresponding well your sample and positive control for detecting phospho EGFR Ser1070 or 100 ul of 200 fold diluted biotinylated anti EGFR to corresponding well your sample help normalize the results of phospho EGFR from different cell lysate being compared for detecting a pan EGFR Incubate for 1 5 hour at room temperature with shaking 5 Discar Discard the solution Repeat the wash as in step 3 6 Add 100 ul of 1x HRP conjugated anti rabbit IgG see Reagent Preparation step 6 to detect rabbit anti phospho EGFR Ser 1070 corresponding well added rabbit anti phospho EGFR or 100 ul of 600 fold diluted HRP Streptavidin to detect biotinylatded anti EGFR antibody corresponding well added HRP Streptavidin Incubate for I 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 9 RayBio Phospho EGFR Ser1070 and pan EGFR ELISA Kit Protocol VIII ASSAY PROCEDURE SUMMARY 1 Prepare all reagents s
11. 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 100 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 5 RayBio Phospho EGFR Ser1070 and pan EGFR ELISA Kit Protocol 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 800 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 See 1 Positive control of part IX TYPICAL DATA for a typical result Dissolve the powder thoroughly by a gentle mix Pipette 300 ul 1x Assay Diluent into each tube Use the Positive Control 1 to produce a dilution series shown below Mix each tube thoroughly before the next transfer 1x Assay
12. unosorbent 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 EGFR 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 EGFR Ser 1070 and pan EGFR help normalize the results of phospho EGFR from different cell lysate being compared in human cell lysate An anti EGFR antibody has been coated onto a 96 well plate Samples are pipetted into the wells and phosphorylated and unphosphorylated EGFR present in a sample is bound to the wells by the immobilized antibody The wells are washed and anti phosphorylated EGFR Ser1070 or biotinylated anti pan EGFR antibody is used to detect phosphorylated or pan EGFR After washing away unbound antibody HRP conjugated anti Rabbit IgG or HRP Streptavidin 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 EGFR Ser1070 or pan EGFR 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 EGFR Ser1070 and pan E
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