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User Manual EGFR Phosphorylation Antibody Array

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1. Note mix tube containing 1 000X HRP Conjugated Streptavidin well before use since precipitation may form during storage V Overview and General Considerations A Preparation of Samples The cell lysate can be prepared as follows For attached cells remove supernatant from cell culture wash cells twice with cold 1 X PBS for suspension cells pellet the cells by RayBio Human EGER Phosphorylation Antibody Array 1 6 spinning down the cells at 1500 rpm for 10 min making sure to remove any remaining PBS before adding Lysis Buffer Solubilize the cells at 2x10 cells ml in 1X Lysis Buffer containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail Set I and Set II Pipette up and down to resuspend cells and rock the lysates gently at 2 8 C for 30 minutes Transfer extracts to microfuge tubes and centrifuge at 14 000 x g for 10 min It is recommended that sample protein concentrations be determined using a total protein assay For incubation with the EGFR Phosphorylation Antibody Array 1 use at a protein concentration of 50 1000 ug ml for cell lysates Lysates should be used immediately or aliquot and stored at 70 C Thawed lysates should be kept on ice prior to use If you experience high background you may further dilute your samples B Handling Array Membranes e Always use forceps to handle membranes and grip the membranes by the edges only e Never allow array membranes to dry during experiments e Avoid
2. Human EGFR Phosphorylation Antibody Array 1 8 10 Note 3 Incubation may be done at room temperature for 2 hours Over night at 4 C Decant the samples from each container and wash 3 times with 2 ml of 1X Wash Buffer I at room temperature with shaking 3 min per wash Carefully remove each array membrane and place all of membranes into a plastic container with a minimum of 20 ml of 1 X Wash Buffer I Rinse the 8 Well Multi dish with deionized or distilled water and dry thoroughly Wash array membranes with 1 X Wash Buffer with shaking Repeat 2 times for a total of 3 washes 5 min per wash Wash 3 times with a minimum of 20 ml of 1X Wash Buffer II at room temperature with shaking 5 min per wash Carefully remove each array membrane from the container return it to the 8 well tray Add 1 ml of diluted Cocktail of Biotin Conjugated Anti EGFR to each membrane Incubate at room temperature with gentle shaking for 2 hours Note Incubation may be done at C for overnight Wash as directed in steps 5 6 and 7 Add 1 5 ml of 1X HRP conjugated streptavidin to each membrane RayBio Human EGFR Phosphorylation Antibody Array 1 9 Note Mix tube containing 1X HRP Conjugated Streptavidin well before use since precipitation may form during storage 11 Incubate at room temperature for 2 hours Note incubation may be done at 4 C for overnight 12 Wash as directed in steps 5 and 6 B Detection Do not let the m
3. 2 Y845 2 average signal intensity of EGFR Tyr845 in array 2 Y845 N2 normalized signal of EGFR Tyr845 in array 2 Y845 N2 Y845 2 EGFR 1 EGFR 2 Antibody affinity to its target varies significantly between antibodies The intensity detected on the array with each antibody depends on this affinity therefore signal intensity comparison can be performed only within the same antibody antigen system and not between different antibodies RayBio Human EGFR Phosphorylation Antibody Array 1 13 EGFR Tyr845 EGFR Tyr845 EGFR EGFR eis Tyr1173 c 4 E Seas EGF treated A431 cells Untreated A431 cells Cell lysate 200 ug ml Cell lysate 200 ug ml Fig 1 Human epidermoid carcinoma cell line A431 cells that were 80 90 confluent were serum starved overnight then exposed to 100 ng ml EGF for 20 minutes at 37 C Control cells were serum starved without the subsequent stimulation with EGF Cell lysates were prepared following the Preparation of Sample portion of our protocol V To use the RayBio Phosphorylation Antibody Array 1 treated or untreated cell lysate was added into antibody array membrane The antibody array membranes were washed and cocktail of biotinylated anti EGFR was used to detect phosphorylated proteins on activated receptors After incubation with HRP Conjugated Streptavidin the signals were visualized by chemiluminescence RayBio Human EGFR Phosphorylation Antibody Array 1 14
4. exposure time eg 5 30 seconds If the signals are too weak increase exposure time eg 5 20 min or overnight Or re incubate membranes overnight with 1X HRP conjugated streptavidin and repeat detection on the second day 4 Save membranes at 20 C to 80 C for future reference VII Interpretation of Results The following figure shows RayBio Human EGFR Phosphorylation Antibody Array I membranes probed with different cell lines The signals were detected by using a chemiluminescence imaging device Alternatively membranes may also be exposed to Kodak X Omat film at room temperature A biotinylated protein provides positive signals indicated Pos on the array map page 15 which can be used to orient the membrane and to normalize the results from different arrays being compared The signals of pan EGER ErbB2 ErbB3 and ErbB4 can also be used to normalize the results of their corresponding phospho proteins if the pan proteins are detectable RayBio Human EGFR Phosphorylation Antibody Array 1 11 One important parameter is the background signal To obtain the best results we suggest that several exposures be attempted We also strongly recommend using a negative control in which the sample is replaced with an appropriate mock buffer according to the array protocol particularly during your first experiment By comparing the signal intensities relative expression levels of target proteins can be made The intensit
5. receptors consists of four different proteins called EGFR ErbB1 HER1 ErbB2 HER2 ErbB3 HER3 and ErbBA HERA Under normal physiological conditions the ErbB receptors play crucial roles in propagating signals regulating cell proliferation differentiation motility and apoptosis EGF receptor family shows clear differences between individual receptors and also a large overlap ErbB1 is the family member with most interaction partners and the highest percentage of tyrosine residues with more than one binding partner ErbB3 is characterized by a large number of binding sites for phosphatidylinositol 3 kinase PI3K while ErbB2 has only few interaction partners with Shc as the most frequent one ErbB1 and ErbB4 have a variety of phosphotyrosines that bind Grb2 or Grb2 and Shc The ErbB1 and ErbB4 have a greater diversity of interaction partners than ErbB2 and ErbB3 ErbB1 and ErbB2 are often over expressed or amplified in cancers making them important targets for drugs currently in use or under development With the RayBio Human EGFR Phosphorylation Antibody Array 1 researchers can now simultaneously detect the relative level of phosphorylation of 17 different specific sites for Human EGFR family in cell lysate By monitoring the changes in protein phosphorylation in your experimental model system you can verify pathway activation in your cell lines without spending excess time and effort in performing an analysis of immunoprecipitation and or W
6. touch Array membrane by hand tips or any sharp tools C Incubation e Completely cover membranes with sample or buffer during incubation and cover eight well tray with lid to avoid drying e Avoid foaming during incubation steps e Perform all incubation and wash steps under gentle rotation RayBio Human EGER Phosphorylation Antibody Array 1 7 e Several incubation steps such as step 4 sample incubation or step 8 biotin Ab incubation or step 10 HRP streptavidin incubation may be done at 4 C for overnight VI Protocol A Blocking and Incubation 1 Place each membrane into the provided 8 well tray top left corner marked with Note The printed side should be facing upward 2 Add 1 ml Blocking Buffer and incubate at room temperature with gentle shaking for 1 hour to block membranes 3 Decant Blocking Buffer from each container Add 1 2 ml of sample into each array membrane and cover with the lid Incubate at room temperature for 2 hours Dilute sample using Blocking Buffer Note 1 We recommended using 1 0 ml of 50 1000 ug ml concentration of cell lysates as starting point we recommended to use a concentration of 200 ug ml of cell lysate Dilute the cell lysates at least 5 folds with Blocking Buffer Note 2 The amount of sample used depends on the abundance of protein More of the sample can be used if signals are too weak If signals are too strong the sample can be diluted further RayBio
7. RayBio Human EGFR Phosphorylation Antibody Array 1 User Manual Revised May 14 2010 For Simultaneously Detecting the Relative Levels of Phosphorylation of EGF Receptors at 17 Different Phosphorylation Sites Cat AAH PER 1 2 AAH PER 1 4 AAH PER 1 8 Please read manual carefully before starting experiment RayBiotech Inc We Provide You with Excellent Protein Array Systems and Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 1 888 547 0580 Website www raybiotech com Email info raybiotech com RayBiotech Inc RayBio Human EGFR Phosphorylation Antibody Array 1 M Protocol TABLE OF CONTENTS I Tif QOL OI esee open eese Eo KENA AMD How It Works K k k II Materials Provided 0 E33 III Additional Materials Required IV Reagent Preparation e V Overview and General Considerations a Preparation of Samples b Handling Array Membrane INCUDANON Xebere MIC sal ne erre E a Blocking and Incubation Di BIS re T SL RNN RNnaRND ID MMDMDMM o l VII Interpretation of Results sese VIII Troubleshooting QuIdG css e ee e IX Reference LAS sc aa ERa RA RR TRER RayBio Human EGFR Phosphorylation Antibody Array 1 1 Y W N oo GO a 1 ON tA 4 SN A Cc I Introduction The EGFR family of membrane
8. RayBio Human EGFR Phosphorylation Antibody Array 1 Map ST EN A HER ERIT HERS TERIN HERE ur E NI IP BES N D 1 r Tj Talak ve EGrR wees EGrn wsez EGER Tyr1045 EGFR 1yr1068 GL a urs LS ee e e TL ET pore armi cem 4 Bank S esser esr mma ERIT ER Jen To eese mwess Ems erri Ems Tyran Xm IE e Esse tmrerr ess mar R Es mrs Eee reas ERES rns pA Ei mer s a Bn eme Neg iic i nnn 8 carm eeee amp mes Ee Bank Bank Nes Bank es EGFR Tyr845 EGFR Tyr1173 Ez N 5 N G y y v o e A xe gt N xw e Na e dP v Qa S S lt e Fig 2 Western blot analysis of extracts from 100 ng ml hEGF treated A431 cells or untreated A431 cells Phospho EGFR Tyr845 or Phospho EGFR Tyr1173 antibodies was used in this assay RayBio Human EGER Phosphorylation Antibody Array 1 15 VIII Troubleshooting Guide Weak signal or no 1 Taking too much time 1 The whole detection process must be signal for Detection completed in 30 min 2 Film developer does 2 Fix film developer not work properly 3 Did not mix HRP 3 Mix tube containing HRP Conjugate streptavidin well before Streptavidin well before use since use precipitates may form during storage 4 Sample is too dilute 4 Increase sample concentration 1 Reduce blocking concentration by diluting in Other SHS 1X Wash Buffer II 2 Slightl
9. embrane dry out during detection The detection process must be completed within 40 minutes without stopping 1 Proceed with detection reaction Add 250 ju of Detection Buffer C and 250 ul of Detection Buffer D for one membrane mix both solutions Drain off excess wash buffer by holding the membrane vertically with forceps Place membrane protein side up mark is on the protein side top left corner on a clean plastic sheet provided in the kit Pipette the mixed Detection Buffer on to the membrane and incubate at room temperature with gentle shaking for 2 minutes Ensure that the detection mixture is completely and evenly covering the membrane without any air bubbles 2 Drain off excess detection reagent by holding the membrane vertically with forceps and touching the edge against a tissue Gently place the membrane protein side up on a piece of plastic sheet mark is on the protein side top left corner Cover the array with another piece of plastic sheet Gently RayBio Human EGFR Phosphorylation Antibody Array 1 10 smooth out any air bubbles Avoid using pressure on the membrane 3 Detect signal directly from membrane using chemiluminescene imaging system or expose to x ray film we recommend to use Kodak X Omat AR film detect signal using film developer Expose the membranes for 40 Seconds Then re expose the film according to the intensity of signals If the signals are too strong background too high reduce
10. estern Blot By using RayBio Human EGFR Phosphorylation antibody Array 1 treated or untreated cell lysate is added into antibody array membranes The antibody array membranes are washed and cocktail of biotin congujated anti EGER is used to detect phosphorylated ErbB1 B4 and pan ErbB1 B4 After incubation with HRP streptavidin the signals are visualized by chemiluminescence RayBio Human EGER Phosphorylation Antibody Array 1 2 Here s how it works SUYO R Incubation of Sample with 2 hrs arrayed antibody chips ke Antibody array chips Cocktail of biotin conjugated anti EGFR A k P Y d 2 hrs Incubation with cocktail of biotin conjugated anti EGFR v Labeled YYY wy Uu IU Data analysis and graph Incubation with labeled 2 hrs Streptavidin lt gt lt e gt lt gt RayBio Human EGFR Phosphorylation Antibody Array 1 3 II Materials Provided Upon receipt the kit should be stored at 20 C After initial use 2X Cell Lysis Buffer Blocking Buffer 20X Wash Buffer I 20X Wash Buffer II Cocktail of Biotin Conjugated Anti EGFR and HRP Conjugated Streptavidin should be stored at 4 C to avoid repeated freeze thaw cycles Array membrane Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail should be kept at 20 C Please use within 6 months from the date of shipment RayBio RayBio Human EGFR Phosphorylation Antibody Array 1 membrane 2 4
11. ies of the signals can be quantified by densitometry Positive controls may be used to normalize the results from different membranes If the pan total EGER ErbB2 ErbB3 or ErbB4 signals are detectable then they may also be used to normalize the signals of their corresponding phospho proteins If desired the phospho protein signals may be normalized to both the positive controls and the pan EGFR signals Normalization of Signals One array should defined as the reference to which the signal intensities of the other arrays should be compared It is up to the researcher to define which array should be the reference The normalization of the array signals to the positive controls may be calculated as follows Pos 1 average signal intensity of positive controls on the reference array Pos 2 average signal intensity of positive controls on Array 2 X 2 signal intensity for a particular spot on Array 2 X N2 the normalized value for that particular spot on Array 2 X N2 X 2 Pos 1 Pos 2 RayBio Human EGFR Phosphorylation Antibody Array 1 12 This calculation may be repeated for the remaining arrays 3 4 5 etc for a particular experiment After positive control normalization normalization of phospho EGER signals to the pan EGFR signals may be calculated according to the following example EGFR 1 average signal intensity of pan EGFR on the reference array EGFR 2 average signal intensity of pan EGFR in array
12. ody production highest quality with very competitive price Monoclonal antibody Recombinant antibody Polyclonal antibody Phase display Antibody angineering Antibody conjugation Recombinant protein production Assay development Antibody array Protein array Peptide array ELISA Phosphorylation assay Tissue array Array printing Contact and non contact arrayers All kinds of substrates of your choice including glass slides membranes and plates RayBio Human EGFR Phosphorylation Antibody Array 1 18 Note RayBio Human EGFR Phosphorylation Antibody Array 1 19 Note RayBio Human EGFR Phosphorylation Antibody Array 1 20 This product is for research use only 2004 RayBiotech Inc RayBio Human EGFR Phosphorylation Antibody Array 1 21
13. or 8 membranes 2X Cell Lysis Buffer 5 ml Protease Inhibitor Cocktail 1 tube for 2 4 membranes and 2 for 8 membranes 100X Phosphatase Inhibitor Cocktail Set I Concentrate 1 tube for 2 4 membranes and 2 for 8 membranes Phosphatase Inhibitor Cocktail Set II 1 tube for 2 4 membranes and 2 for 8 membranes Blocking Buffer 25 ml for less 4 membranes and 50 ml for 8 membranes 20X Wash Buffer I 30 ml 20X Wash Buffer II 30 ml Cocktail of Buiotin Conjugated Anti EGFR 1 tube for 2 membranes 2 for 4 membranes and 4 for 8 membranes 1 000X HRP Conjugated Streptavidin 18 ul Detection Buffer C 1 5 ml for 2 4 membranes 2 5 ml for 8 membranes Detection Buffer D 1 5 ml for 2 4 membranes 2 5 ml for 8 membranes Eight Well Tray 1 each Human EGFR Phosphorylation Antibody Array 1 4 e Plastic sheets III Additional Materials Required Small plastic boxes or containers Shaker Plastic sheet protector or Saran Wrap Kodak X Omat AR film REF 165 1454 and film processor or Chemiluminescence imaging system IV Reagent Preparation 1 Protease Inhibitor Cocktail Briefly spin down the Protease Inhibitor Cocktail tube before use Add 60 ul of 1X Lysis Buffer into the vial to prepare a 100X Protease Inhibitor Cocktail Concentrate 2 Phosphatase Inhibitor Cocktail Set II Briefly spin down the Phosphatase Inhibitor Cocktail Set II tube before use Add 180 ul of 1X Lysis Buffer into each vial to prepare 25X Phospha
14. r 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 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 Assay service just simply send your samples and get data in 1 to 2 weeks Antibody array Protein array ELISA Quantibody array Antib
15. tase Inhibitor Cocktail Set II Concentrate Dissolve the powder thoroughly by a gentle mix 3 2X Cell Lysis Buffer Cell lysis buffer should be diluted 2 fold with deionized or distilled water before use Add 20 ul of prepared 100X Protease Inhibitor Cocktail Concentrate and 20 ul of 100X Phosphatase Inhibitor Cocktail Set I Concentrate bring Set I concentrate tube to room temperature to thaw the solution before use and 80 ul Phosphatase Inhibitor Cocktail Set II into 1 9 ml 1X Lysis Buffer before use Mix well RayBio Human EGFR Phosphorylation Antibody Array 1 5 4 20X Washing Buffer I or II If the Wash Buffer Concentrate 20X contains visible crystals warm to room temperature and mix gently until dissolved Dilute 25 ml of Wash Buffer Concentrate into deionized or distilled water to yield 500 ml of 1 X Wash Buffer Cocktail of Biotin Conjugated Anti EGFR Briefly spin the Detection Antibody tube before use Add 100 ul of Blocking Buffer to the tube Mix gently and transfer all mixture to a tube containing 2 1 ml of Blocking Buffer to prepare 1X Cocktail of Biotin Conjugated Anti EGFR 1000X HRP Conjugated Streptavidin briefly spin down the HRP Streptavidin Concentrate and pipette up and down to mix gently before use E g add 5 ul of HRP Conjugated Streptavidin concentrate into a tube with 5 ml Blocking Buffer Mix gently to prepare 1X HRP Conjugated Streptavidin don t store the diluted Streptavidin for next day use
16. y increase HRP concentrations 3 Slightly increase biotinylate antibody concentrations 4 Expose film for overnight to detect weak signal Uneven signal 1 Bubbles formed 1 Remove bubbles during incubation during incubation 2 Membranes were not 2 Completely cover membranes with solution completely covered by solution High background 1 Exposure to x ray file 1 Decrease exposure time is too long 2 Membranes were 2 Completely cover membranes with solution allowed to dry out during during experiment experiment 3 Sample is too 3 Use more diluted sample concentrated RayBio Human EGFR Phosphorylation Antibody Array 1 16 IX Reference List 1 Holbro T Hynes NE 2004 ErbB receptors directing key signaling networks throughout life Annu Rev Pharmacol Toxicol 44 195 217 2 Marmor MD Skaria KB Yarden Y 2004 Signal transduction and oncogenesis by ErbB HER receptors Int J Radiat Oncol Biol Phys 58 903 913 3 Huang RP Huang R Fan Y and Lin Y 2001 A novel method for high throughput protein profiling from conditioned media and patient s sera Ana Biochem 294 1 55 62 4 Huang R Lin Y Wang CC J et al 2002 Connexin suppresses human glioblastoma cell growth by down regulation of monocyte chemotactic protein 1 as discovered using protein array technology Cancer Res 62 2806 2812 RayBio Human EGFR Phosphorylation Antibody Array 1 17 RayBiotech Inc the protein array pionee

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