User Manual RTK Phosphorylation Antibody Array (Membranes)
Contents
1. OA A WN BW Wb on n Aa e I Introduction Protein phosphorylation plays an unusually prominent role in cell signaling development and growth The RayBio Phosphorylation Antibody Array G series I is a very rapid convenient and sensitive assay to simultaneous detect multiple protein phosphorylations and can be used to monitor activation or function of important biological pathways RayBiotech is committed to developing a series of phosphorylation antibody arrays Our first product in this series is RayBio Phosphorylation Antibody Array I which is specifically designed for simultaneously identifying the relative levels of phosphorylation of 71 different human receptor tyrosine kinases RTKs in cell lysates By monitoring the changes in protein tyrosine 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 Western Blot By using RayBio Phosphorylation antibody array I treated or untreated cell lysate is added into antibody array membranes The antibody array membranes are washed and biotinylated anti phosphotyrosine antibody is used to detect phosphorylated tyrosines on activated receptors After incubation with HRP streptavidin the signals are visualized by chemiluminescence RayBio Phosphorylation Antibody Array I 2 Here s how it works YYY Y ve Incubation of2. 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 RayBio Phosphorylation Antibody Array I 11 By comparing the signal intensities relative expression levels of target proteins can be made The intensities of signals can be quantified by densitometry Positive control can be used to normalize the results from different membranes being compared 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 LILET E ErbB3 pork CS ErbB2 Untreated A431 cells EGF treated 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 10 minutes at 37 C Control cells were serum starved without the subsequent stimulation with EGF Cell lysates were prepared following the Sample Preparation portion of our protocol IV To wuse the RayBio Phosphorylation Antibody Array I treated or untreated cell lysa
3. Sample with 2 hrs arrayed antibody chips Biotinylated Anti Incubation with 2 hrs phosphotyrosine y A lt Biotinylated Y l y Y y Antrphosphotyrosine y Y Y Data analysis and graph Antibody array chips Labeled Streptavidin Incubation with labeled 2 hrs Streptavidin lt a0 RayBio Phosphorylation Antibody Array I 3 II Materials Provided Upon receipt the kit should be stored at 20 C to 80 C Please use within 6 months from the date of shipment After initial use 2X Cell Lysis Buffer Blocking Buffer 20X Wash Buffer I 20X Wash Buffer II Buiotin Conjugated Anti phosphotyrosine and HRP Conjugated Streptavidin should be stored at 4 C to avoid repeated freeze thaw cycles Array I membrane Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail should be kept at 20 to 80 C Use within three months after initial use e RayBio Phosphorylation Antibody Array I membrane 2 4 or 8 membranes e 2X Cell Lysis Buffer 5 ml e Protease Inhibitor Cocktail 1 tube for 2 4 membranes and 2 for 8 membranes e Phosphatase Inhibitor Cocktail 1 tube for 2 4 membranes and 2 for 8 membranes e Blocking Buffer 25 ml for less 4 membranes and 50 ml for 8 membranes e 20X Wash Buffer I 30 ml e 20X Wash Buffer II 30 ml e Biotin Conjugated Anti phosphotyrosine 1 tube for 2 membranes 2 for 4 membranes and 4 for 8 membranes e 000X HRP Conjugated S
4. clinical diagnosis Our produces may not be resold modified for resale or used to manufacture commercial products without written approval by RayBiotech Inc Under no circumstances shall RayBiotech be liable for any damages arising out of the use of the materials Products are guaranteed for 6 months from the date of purchase when handled and stored properly In the event of any defect in quality or merchantability RayBiotech s liability to buyer for any claim relating to products shall be limited to replacement or refund of the purchase price ECL is a trademark of Amersham Pharmacia Biotech X Omat 1s a trademark of Kodak RayBio Phosphorylation Antibody Array I 16 This product is for research use only 2004 RayBiotech Inc RayBio Phosphorylation Antibody Array I 17
5. 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 Phosphorylation Antibody Array I 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 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 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 RayBio Phosphorylation Antibody Array I 7 VI Protocol A Blocking and Incubation 1 Place each membrane into the provided 8 well tray mark 1s on the antibody printed side Note The printed side should be facing upward 2 Add 1 ml Blocking Buffer and incubate at room temperature for hour to block membranes 3 D
6. RayBio Phosphorylation Antibody Array I For Simultaneously Detecting the Relative Level of Tyrosine Phosphorylation of Human Receptor Tyrosine Kinases RTKs User Manual Revised Jul 25 2007 Cat AAH PRTK 1 2 AAH PRTK 1 4 AAH PRTK 1 8 1 es RayBiotech Inc We Provide You With Excellent Protein Array System And Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 1 888 547 0580 www raybiotech com Email info raybiotech com RayBiotech Inc RayBio Phosphorylation Antibody Array I Protocol TABLE OF CONTENTS J ntroduction ccc ccc eee eee eee ee ee eeaens How It WorkS ccc ccc cece cee eee e eee eeeeenees II Materials Provided ccc cece ce cece ee ee n IHI Additional Materials Required IV Reagent Preparation ccc cece cece eens V Overview and General Considerations a Preparation of Samples cee b Handling Array Membrane c Incubation ccc cece cece cee e cece ee eee eees VI PEOUOCOlis ssendeuteosuehcdeacarertancasetehouaeacaaeess a Blocking and Incubation 45 D Detection ccc cece cece EE VII Interpretation of Results 00 cece eee ees VIII Troubleshooting Guide 0 cece eee IX Reference List 0 cece cece eect eee e cease RayBio Phosphorylation Antibody Array I 1 COON NA
7. and Tempst P MolCell Proteomics 2004 3 1102 1118 Analysis of receptor signaling pathways by mass spectrometry Identification of Vav 2 as a substrate of the epidermal and platelet derived growth factor receptors Pandey A Podtelejnikov AV Blagoev B Bustelo XR Mann M and Lodish HF PNAS 2000 97 1 179 184 Reduced T cell and dendritic cell function 1s related to cyclooxygenase 2 overexpression and protaglandin e 2 secretion in patients with breast cancer Pockaj BA Basu GD Annal Surg Oncol 2004 3 327 344 Cytokine Antibody Arrays A Promising Tool to Identify Molecular Targets for Drug Discovery Huang RP Comb Chem High Throughput Screen 2003 6 79 99 Connexin suppresses human glioblastoma cell growth by down regulation of monocyte chemotactic protein 1 as discovered using protein array technology Huang R Lin Y Wang CC J et al Cancer Res 2002 62 2806 28 12 RayBio Phosphorylation Antibody Array I 15 7 Profiling of cytokine expression by biotin labeled based protein arrays Lin Y Huang R Chen L P et al Proteomics 2003 3 1750 1757 8 A novel method for high throughput protein profiling from conditioned media and patient s sera Huang RP Huang R Fan Y and Lin Y Ana Biochem 2001 294 1 55 62 RayBio is the trademark of RayBiotech Inc Cytokine protein arrays are RayBiotech patent pending technology This product is intended for research only and is not to be used for
8. ecant 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 2 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 fold 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 Note 3 Incubation may be done at room temperature for 2 hours Over night at 4 C RayBio Phosphorylation Antibody Array I g 4 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 5 Carefully remove each array membrane and place all of membranes into a plastic container with a minimum of 20 ml of 1X Wash Buffer I Rinse the 8 Well Multi dish with deionized or distilled water and dry thoroughly Wash array membranes with 1X Wash Buffer with shaking Repeat 2 times for a total of 3 washes 5 min per wash 6 Wash 3 times with a minimum of 20 ml of 1X Wash Buffer II at room temperature with shaking 5 min per wash 7 Carefully remove each array membrane from the container return it to the 8 well tray 8 Add 1 2 ml of diluted biotin c
9. is on the protein side top left corner Cover the array with another piece of plastic sheet Gently 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 RayBio Phosphorylation Antibody Array I 10 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 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 Because the spots are very small you may need to magnify signals after scanning your films If the signals are too weak or can t be seen in your films 4 Save membranes at 20 C to 80 C for future reference VII Interpretation of Results The following figure shows RayBio Phosphorylation Antibody Array I membranes probed with different cell lines The signals were detected by using a chemiluminescene imaging device Membranes also can be exposed to Kodak X Omat film at room temperature A biotinylated protein provides positive signals which can be used to identify the orientation and to normalize the results from different wells being compared
10. of 1X Wash Buffer RayBio Phosphorylation Antibody Array I 5 5 Biotinylated Anti phosphotyrosine 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 5 ml of Blocking Buffer to prepare 1X Biotinylated Anti phosphotyrosine 6 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 Streptavin for next day use 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 1X PBS for suspension cells pellet the cells by 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 Pipette up and down to resuspend cells and rock the lysates gently at 2 8 C RayBio Phosphorylation Antibody Array I 6 for 30 minutes Transfer extracts to
11. ole 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 1X Wash Buffer II 5 Other 2 Slightly 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 Phosphorylation Antibody Array I 14 IX Reference List l Profiling receptor tyrosine kinase activation by using Ab microarrays Nielsen UB Cardone MH Sinskey AJ MacBeath G and Sorger PK PNAS 2003 100 16 9330 9335 A Prototype Antibody Microarray Platform to Monitor Changes in Protein Tyrosine Phosphorylation Gembitsky DS Lawlor K Jacovina A Yaneva M
12. onjugated antibodies to each membrane Incubate at room temperature for 2 hours Note Incubation may be done at 4 C for overnight 9 Wash as directed in steps 5 6 and 7 10 Add 1 5 ml of IX HRP conjugated streptavidin to each membrane 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 RayBio Phosphorylation Antibody Array I 9 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 membrane dry out during detection The detection process must be completed within 40 minutes without stopping 1 Proceed with detection reaction Add 250 ul of Detection Buffer C and 250 ul of Detection Buffer D for one membrane m x 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 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
13. te was added into antibody array membrane The antibody array membranes were washed and biotinylated anti phosphotyrosine antibody was used to detect phosphorylated tyrosines on activated receptors After incubation with HRP Conjugated Streptavidin the signals were visualized by chemiluminescence RayBio Phosphorylation Antibody Array I 12 Untreated Treated Untreated Treated Untreated Treated EGFR ErbB2 ErbB3 Fig 2 Immunoprecipitations were done using anti EGFR ErbB2 and ErbB3 monoclonal antibodies and Protein A Immunoblots were incubated with a biotinylated anti phosphotyrosine monoclonal antibody to detect phosphorylated taget protein receptors Bands were visualized with Streptavidin HRP followed by chemiluminescent detection substrate RayBio Phosphorylation Antibody Array Map 2 NEG NEG NEG NEG Axl Axl Bik Blk BMX BMX Btk Btk 3 4 EphA4 EphA4 EphA5 EphA5 EphA6 EphA6 EphA7 EphA7 EphA8 EphA8 EphB1 EphB1 5 6 ErbB4 ErbB4 FAK FAK FER FER FGFR1 FGFR1 FGFR2 FGFR2 FGFR2 FGFR2 a isoform a isoform 7 R x 8 Insulin R Insulin R Itk Itk JAK1 JAK1 JAK2 JAK2 JAK3 JAK3 LCK LCK 9 LK LK Lyn Ly MATK MATK M CSFR M CSFR MUSK MUSK NGFR NGFR 10 PDGFR a PDGFR a PDGFR B PDGFR B PYK2 PYK2 RET RET ROR1 ROR1 ROR2 ROR2 11 12 Tie 1 Tie 1 Tie 2 Tie 2 TNK1 TNK1 TRKB TRKB TXK TXK NEG NEG RayBio Phosphorylation Antibody Array I 13 VIII Troubleshooting Guide Weak signal or no 1 Taking too much time 1 The wh
14. treptavidin 18 ul e Detection Buffer C 1 5 ml for 2 4 membranes 2 5 ml for 8 membranes e Detection Buffer D 1 5 ml for 2 membranes 2 5 ml for 8 membranes e Fight Well Tray 1 each e Plastic sheets RayBio Phosphorylation Antibody Array I 4 III Additional Materials Required Small plastic boxes or containers Orbital shaker Plastic sheet protector or Saran Wrap Kodak X Omat AR film CREF 165 1454 and film processor or Chemiluminescence imaging system IV Reagent Preparation l 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 Briefly spin down the Phosphatase Inhibitor Cocktail tube before use Add 180 ul of 1X Lysis Buffer into the vial to prepare 25X Phosphatase Inhibitor Cocktail 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 Add 20 ul Protease Inhibitor Cocktail Concentrate and 80 ul Phosphatase Inhibitor Cocktail Concentrate into 1 9 ml 1X Lysis Buffer before use Mix well 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
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