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1. 2 0 OD 450 nm 5 0 5 0 0 Phospho EGFR Tyr 1086 EGFR Western Blot 0 10 Anti phospho EGFR Anti EGFR Tyr1086 11 RayBio Phospho EGFR Tyr 1086 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 Tyr 1086 ELISA 3 5 3 0 2 5 2 0 1 5 1 0 0 5 0 0 450 nm OD 5 1 0 2 0 04 0 08 ug Western Blot 12 RayBio Phospho EGFR Tyr 1086 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 13 RayBio Phospho EGFR Tyr 1086 and pan EGFR ELISA Kit Protocol XI TROUBLESHOOTING GUIDE washed Contaminated wash buffer 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 backgrou
2. 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 10 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 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 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 5 RayBio Phospho EGFR Tyr 1086 and pan EGFR ELISA Kit Protocol Solution See 1 Positive control of part IX TYPICAL DATA for a typical result Dissolve the powder thoroughly by a gentle mix it can be removed by centrifuge if any precipitate in the solution is found Pipette 30
3. Phospho EGFR Tyr 1086 and pan EGFR ELISA Kit Protocol D 1 Assay Diluent Item E2 HRP Streptavidin Item G TMB One Step Substrate Reagent Item H Stop Solution Item I Cell Lysate Buffer Item J and Biotinylated Antibody 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 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 I liter graduated cylinders Distilled or deionized water Tubes to prepare sample dilutions AHAAQYNNANB 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 4 RayBio Phospho EGFR Tyr 1086 and pan EGFR ELISA Kit Protocol
4. 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 primary antibody 1X Anti phospho EGFR Tyr 1086 Reagent Preparation step 7 or 1X Biotinylated Anti EGFR Reagent Preparation step 7 to each well Incubate for 1 5 hour at room temperature with shaking Discard Discard the solution Repeat the wash as in step 3 Add 100 ul 1x HRP conjugated anti rabbit IgG see Reagent Preparation step 6 to detect rabbit anti phospho EGFR Tyr1086 corresponding well added rabbit phospho EGFR antibody or 100 ul of 600 diluted HRP Streptavidin to detect biotinylatded anti EGFR corresponding well added 8 RayBio Phospho EGFR Tyr 1086 and pan EGFR ELISA Kit Protocol biotinylatded anti EGFR antibody 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 VIII ASSAY PROCEDURE SUMMARY 1 Prepare all reagents samples and standards as instructed 2 Add 100 ul
5. 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 J 4 Add 100 ul prepared secondary antibody solution Incubate hour at room temperature 9 RayBio Phospho EGFR Tyr 1086 and pan EGFR ELISA Kit Protocol 5 Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature U 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 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 OD 450 nm 0 1 T T T 7 T P 1 P 2 P 3 P 4 P 5 Positive control dilution series 10 RayBio Phospho EGFR Tyr 1086 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 3 0 mm Untreated A431 a2 EGF treated A431
6. 0 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 Diluent serves as the background 150ul Positive Control Stock Solution 300 ul 150ul 150 ul 150 ul 150 pl 1x Assay Diluent fa Om 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 anti phospho EGFR Tyr 1086 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 RayBio Phospho EGFR Tyr 1086 and pan EGFR 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 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 t
7. RayBio Phospho EGFR Tyr 1086 and Pan EGFR ELISA Kit For Measuring Phospho EGFR Tyr 1086 and Pan EGFR in Human Cell Lysates User Manual Revised Mar 1 2012 RayBio EGFR Tyr 1086 and Pan EGFR ELISA Kit Protocol Cat PEL EGFR Y 1086 T 2 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 Tyr 1086 and Pan EGFR ELISA Kit Protocol TABLE OF CONTENTS I INMOGUCHOM 224 ccscecersuctesievetwxsGaussusyeereareeds 2 H Material Provided iiss ccecoseesiuwasucwedcsesindesrees 3 MI Pt 3 IV Additional Materials Required 04 4 V Sample Preparative cease ties acxndvarensasdeadeeeas 4 VI Reagent Preparation 00 cece cece e ee eens 5 VIL Assay Procedute smsssshresinesssaenenedanesdkvnke 8 VIII Assay Procedure Summa ry 000cee eee 9 IX Typical Da sees seeriessersccneeoeraerctenasvesepeereeneeemeces 10 i Positive Contr l ugussvsiraemsutdvonsaen vad 10 ii Recombinant Human EGF Stimulation of A431 Cell RAMS inc sep cosvaseavincciedantornasuatsedec 11 iii SND 12 X ARGTTENCCS eniehrass ci iebieamsieutabiasveorsaseierseees 13 XI Troubleshooting Guide cece cece eeeees 14 1 RayBio Phospho EGFR Tyr 1086 and pan EGFR ELISA Kit Protocol I INTRODUCTION R
8. at 450 nm 2 RayBio Phospho EGFR Tyr 1086 and pan EGFR ELISA Kit Protocol I MATERIAL PROVIDED 1 2 8 9 10 11 EGFR Microplate Item A 96 wells 12 strips x 8 wells coated with monoclonal anti EGFR Wash Buffer Concentrate 20x Item B 25 ml of 20x concentrated solution Anti phospho EGFR Tyr 1086 Item C Ivial rabbit anti EGFR Tyr 1086 HRP conjugated Anti rabbit IgG Item D 1 25 ul of 500x concentrated HRP conjugated anti rabbit IgG Biotinylated Anti EGFR Item L 1 vial goat anti 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 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 Lysate Buffer not including protease and phosphatase inhibitors Positive Control A431S001 1 Item K 1 vial of lyophilized powder from A431 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 HRP conjugated Anti rabbit IgG Item RayBio 3
9. ayBio Phospho EGFR Tyr 1086 and Pan EGFR 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 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 human phospho EGFR Tyr 1086 and Pan EGFR help normalize the results of phospho EGFR from different cell lysate being compared An anti EGER 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 Tyr 1086 or anti pan EGFR antibody is used to detect phosphorylated or non phosphorylated 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 Tyr1086 or pan EGFR bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured
10. dy 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 Antibody array Protein array Peptide array ELISA Phosphorylation assay 15 RayBio Phospho EGFR Tyr 1086 and pan EGFR 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 16 RayBio Phospho EGFR Tyr 1086 and pan EGFR ELISA Kit Protocol This product is for research use
11. nd a Plate is insufficiently 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 well before measurement and read OD immediately c Ensure correct dilution 14 RayBio Phospho EGFR Tyr 1086 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 detection up to 200 proteins cytokine chemokine growth factor adipokine angiogenic factor protease in one experiment Phosphorylation Antibo
12. o 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 be diluted 200 folds with 1x Assay Diluent and used in step 4 of Part VI Assay Procedure 8 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 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 7 RayBio Phospho EGFR Tyr 1086 and pan EGFR ELISA Kit Protocol VIL 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 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
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