User Manual RTK Phosphorylation Antibody Array (Glass)
Contents
1. 20X Wash Buffer I 30ml 20X Wash Buffer IT 30ml Biotin Conjugated Anti phosphotyrosine 1 2 tubes 1 tube for 4 subarrary chips and 2 for 8 subarray chips Alexa Fluor 555 Conjugated Streptavidin 1 2 tubes 1 tube for 4 subarrary chips and 2 for 8 subarray chips Wash Buffer III 20 ml Adhesive film 4 RayBio Human RTK Phosphorylation Antibody Array G series 1 Protocol III Additional Materials Required Orbital shaker Laser scanner for fluorescence detection Aluminum foil Distilled water Plastic box 50 ml Centrifuge tube Isopropanol 2 propanol Layout of Array Glass Chip Array LILILILI LLM Blank gt OOL Barcode 4 arrays in one glass chip 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 5 RayBio Human RTK Phosphorylation Antibody Array G series 1 Protocol 2 Phosphatase Inhibitor Cocktail Briefly spin down the GO 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 2X Cell Lysis Buffer Cell lysis buffer should be diluted 2 fold with deionized or distilled water Add 20 ul of Protease Inhibitor Cocktail Concentrate and 80 ul of Phosphatase Inhibito2. Introduction Protein phosphorylation plays an unusually prominent role in cell signaling development and growth The RayBio Human RTK Phosphorylation Antibody Array G series 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 Human RTK Phosphorylation Antibody Array which is specifically designed for simultaneously identifying the relative levels of phosphorylation of 71 different Human Receptor Tyrosine Kinases RTKs in cell lysate 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 To use the RayBio Human RTK Phosphorylation Antibody Array G series 1 treated or untreated cell lysate is added into antibody array glass chip The antibody array chips are washed and biotinylated anti phosphotyrosine antibodies are used to detect phosphorylated tyrosines on activated receptors After incubation with Alexa Fluor 555 Conjugated Streptavidin image the signals using laser scanner such as the Axon GenePix using the cy3 channel 2 RayBio Human RTK Phosphorylation Antibo
3. VII Interpretation of Results The following figure shows the RayBio Human RTK Phosphorylation Antibody Array probed with different cell lysates The images were captured using laser scanner A biotinylated protein produces positive control signals which can be used to identify the orientation and to normalize the results from different wells being compared Antibody affinity to its target varies significantly between antibodies The fluorescence 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 Untreated A431 cells EGF treated A431 cells Cell lysate 400 ug ml Cell lysate 400 ug ml Fi Z 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 use the RayBio Human RTK Phosphorylation Antibody Array G series 1 treated or untreated cell lysate was added into antibody array glass chips The antibody array 14 RayBio Human RTK Phosphorylation Antibody Array G series 1 Protocol chips were washed and biotinylated anti phosphotyrosine antibody was used to detect phosphorylated tyrosines on acti
4. down the cells at 1500 rpm for 10 min making sure to remove any remaining PBS before adding Lysis Buffer containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail Solubilize the cells at 2x10 cells ml in 1X Lysis Buffer Pipette up and down to resuspend the cells and rock the lysates gently at 2 8 C for 30 minutes Transfer extracts to microcentrifuge tubes and centrifuge at 14 000 x g for 5 min It is recommended that sample protein concentrations be determined using a total protein assay For incubation with the Phosphorylation Antibody Array I use cell lysates in 50 1000 ug ml of concentration as starting point we recommended to use 400 ug ml of cell lysate dilute the lysate at least 5 fold with Blocking Buffer Lysates should be used immediately or aliquoted 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 sample If signals are too week the cell lysates can be pretreated by immunoprecipitations before incubation with array membranes Immunoprecipitations can be done using anti phosphotyrosine and protein A 7 RayBio Human RTK Phosphorylation Antibody Array G series Protocol B Handling glass chips The microarray slides are sensitive do not touch the array surface by tips forceps or hand Hold the slides by the edges only Handle all buffers and slides with latex free gloves Avoid breaking glass slide
5. incubation chamber barcode facing upward as in step 1 2 Gently snap one edge of a snap on side as shown in step 2 3 Gently press other of side against lab bench and push in Direction shown in step 3 4 Repeat with the other side 12 RayBio Human RTK Phosphorylation Antibody Array G series Protocol C Fluorescence Detection 1 Put the glass chip into a 50 ml centrifuge tube dry the glass chip by centrifuge at 1 000 rpm for 3 minutes Or dry the glass chip by a compressed N stream Or let glass chip dry completely in clean air condition protect from light Make sure the slides are absolutely dry before the scanning procedure Image the signals using laser scanner such as the Axon GenePix using the cy3 channel Note We recommend scanning slides right after experiment You also can store the slide at 20 C in dark for several days Jf you do not have a laser scanner we can provide service for you Just simply send your slide to us and we will take care of it Note Put the glass chip into a tube with 40 ml of 30 Wash Buffer Il in isopropanol add 15 ml of Wash Buffer III into a tube with 35 ml of isopropanol mix well and incubate for 10 min at room temperature if the background is not even or too high cover the tube with aluminum foil to avoid exposure to light or incubate in dark room Dry the slide and re scan the slide 13 RayBio Human RTK Phosphorylation Antibody Array G series 1 Protocol
6. 118 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 17 RayBio Human RTK Phosphorylation Antibody Array G series 1 Protocol 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 18 RayBio Human RTK Phosphorylation Antibody Array G series 1 Protocol RayBiotech Inc the protein array pioneer company strives to research and develop new products to meet demands of the biomedic
7. Clean environment Incubation Completely cover array area with sample or buffer during incubation and cover the incubation chamber with adhesive film or plastic sheet protector to avoid drying Avoid foaming during incubation steps Perform all incubation and wash steps under gentle rotation Cover the incubation chamber with adhesive film during incubation particularly when incubation is more than 2 hours or less than 50 ul of sample or reagent is used Avoid cross contamination from overflowing solution to neighboring wells Several incubation steps such as step 3 sample incubation step 8 biotin Ab incubation or step 11 Alexa Fluor 555 streptavidin incubation may be done at 4 C for overnight Please make sure to cover the incubation chamber tightly to prevent evaporation 8 RayBio Human RTK Phosphorylation Antibody Array G series 1 Protocol VI Protocol A Dry the Glass Chip Open the box containing glass chip with frame and take it out and then let it air dry for 1 hour in clean environment before use Note Protect the chip from dust or others contaminants B Blocking and Incubation 1 Add 100 ul of 1 X Blocking Buffer into each well and incubate at room temperature for 30 min to block slides Make sure no bubbles are in the well Note Only add reagents to wells printed with antibodies 2 Decant Blocking Buffer from each well make sure to remove all of buffer Add 100 ul of each sample into appro
8. RayBio Human RTK Phosphorylation Antibody Array G series 1 For Simultaneously Detecting the Relative Level of Tyrosin Phosphorylation of Human Receptor Tyrosine Kinases RTKs User Manual Revised Nov 15 2007 Cat AAH PRTK G1 4 and AAH PRTK G1 8 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 Web www raybiotech com Email info raybiotech com RayBiotech Inc RayBio Huamn RTK Phosphorylation Antibody Array G series 1 Protocol TABLE OF CONTENTS I Intoduct on 2 How HUW 6 e AE YE EEE POND 3 II MaternalsProvided 4 IHI Additional Materials Required 5 IV ReagentPreparatlon 5 V Overview and General Considerations 7 A PreparattonofSamples 7 B HandlingGlassChips 7 C incubatilon 8 VE 2 OLOCOLs cco N EE EE EE 9 A D ytheArrayChips 9 B Blockingandincubatlon 9 C FluorescenceDetecdon 13 VII MmterpretatonotfResulis 14 VilTroubleshoot ngGuide 16 IX ELER S ba sne e e e b ek a e b 17 I RayBio Human RTK Phosphorylation Antibody Array G series Protocol I
9. al 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 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 Antibody production highest quality with very competitive price Monoclonal antibody Recomb
10. dy Array G series 1 Protocol Here s how it works YYYYY ve Antibody array chips Incubation of Sample with 2 hrs arrayed antibody chips A Biotinylated anti a7 lt 7 Sik Phosphotyrosine gt Incubation with 2 hrs Biotinylated anti Phosphotyrosine Y lt Labeled Streptavidin 4 Incubation with labeled 2 hrs Streptavidin Data analysis and graph 3 RayBio Human RTK Phosphorylation Antibody Array G series Protocol 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 Biotin Conjugated Anti phosphotyrosine and Alexa Fluor 555 Conjugated Streptavidin should be stored at 4 C to avoid repeated freeze thaw cycles Array I Glass Chip Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail should be kept at 20 9 to 80 C Use within three months after initial use RayBio Human RTK Phosphorylation Antibody Array G series Glass Chip with Frame each slide with 4 Subarrays slide for 4 subarray chips and 2 for 8 subarray chips 2X Cell Lysis Buffer 5 ml Protease Inhibitor Cocktail 1 2 tubes 1 tube for 4 subarrary chips and 2 for 8 subarray chips Phosphatase Inhibitor Cocktail 1 2 tubes 1 tube for 4 subarrary chips and 2 for 8 subarray chips Blocking Buffer 8 ml
11. eries 1 Protocol VIII Troubleshooting Guide Problem Cause Recommendation Weak signal Inadequate detection Check laser power and PMT parameters Inadequate reagent volumes Check pipettors and or improper dilution ensure correct preparation Short incubation times Ensure sufficient incubation time and change sample incubation step to overnight Too low protein concentration Don t make too low dilution in sample Or concentrate sample Improper storage of kit Store kit at suggested temperature High background Excess of biotinylated antibodies Make sure to use the correct amount of antibodies Excess of streptavidin Make sure to use the correct amount of streptavidin Inadequate detection Check laser power and PMT parameters Inadequate wash Increase the volume of wash buffer and incubation time Uneven signal Bubbles formed during Avoid bubble formation incubation during incubation Arrays are not completely Completely cover arrays covered by reagent with solution 16 RayBio Human RTK Phosphorylation Antibody Array G series 1 Protocol 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 n Protein Tyrosine Phosphorylation Gembitsky DS Lawlor K Jacovina A Yaneva M and Tempst P Mol Cell Proteomics 2004 3 1102 1
12. inant 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 19 RayBio Human RTK Phosphorylation Antibody Array G series 1 Protocol Note 20 RayBio Human RTK Phosphorylation Antibody Array G series Protocol This product is for research use only 2004 RayBiotech Inc 21 RayBio Human RTK Phosphorylation Antibody Array G series Protocol
13. n glass chip with 40 ml of 1X Wash Buffer II in 1 min 7 Remove all of Wash Buffer II in the well Add 100 ul of 1x Biotin conjugated anti Phosphotyrosine to each corresponding well Incubate at room temperature for 2 hours 10 RayBio Human RTK Phosphorylation Antibody Array G series 1 Protocol 8 Decant the antibody solution and wash as directed in steps 4 5 6 and 7 9 Add 100 ul of IX Alexa Fluor 555 conjugated streptavidin to each subarray Cover the incubation chamber with Adhesive film Cover the plate with aluminum foil to avoid exposure to light or incubate in dark room 10 Incubate at room temperature for 2 hours Note Incubation may be done at 4 C for overnight 11 Decant the antibody solution and disassemble the slide out of the incubation frame and chamber 12 Gently put the glass chip into a 50 ml centrifuge tube or a plastic box with 40 ml of IX Wash Buffer I Gently roll over the tube or shake for 5 min Remove the wash buffer Repeat 2 times for a total of 3 washes 11 RayBio Human RTK Phosphorylation Antibody Array G series Protocol 13 Wash the glass chip with 40 ml of Wash Buffer II Repeat one time for a total of two washes 5 min per wash 14 Finally wash the glass chip with 40 ml of deionized or distilled water Note You may assemble the glass chip into an incubation chamber by following step You may want to practice assembling the device with a blank glass slide 1 Apply slide to
14. priate wells Incubate arrays with sample at room temperature for 2 hours or 4 C for overnight Note We recommended using 100 ul of cell lysates in 50 1000 ug ml of concentration as starting point we recommended to use 400 ug ml of concentration of cell lysate Dilute the lysate at least 5 fold with blocking buffer Make sure there is no bubble in the wells Note The amount of sample used depends on the abundance of target proteins More sample can be used if signals are 9 RayBio Human RTK Phosphorylation Antibody Array G series 1 Protocol too weak If signals are too strong the sample can be diluted further Note Incubation may be done at 4 C overnight Note The cell lysates can be pretreated by immunoprecipitations before incubation with array membranes if signals are too week Immunoprecipitations can be done using anti phosphotyrosine and protein A The elution samples from protein A can be diluted with Blocking Buffer and then incubate with array membranes 3 Decant the samples from each well and wash 3 times with 100 ul of 1X Wash Buffer I at room temperature with gentle shaking 5 min per wash Note avoid solution flowing into neighboring wells 4 Slowly rinse the wells in glass chip with 40 ml of IX Wash Buffer I n 1 min 5 Decant the Wash Buffer I from each well and wash 3 times with 100 ul of IX Wash Buffer II at room temperature with gentle shaking 5 min per wash 6 Slowly rinse the wells i
15. r Cocktail Concentrate into 1 9 ml of IX Lysis Buffer before use Mix well 20X Washing Buffer I or II If the 20X Wash Concentrate 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 1X Wash Buffer Biotinylated anti Phosphotyrosine Briefly spin the Detection Antibody tube before use Add 65 ul of Blocking Buffer into the tube to prepare a Biotinylated Anti phosphotyrosine Concentrate Pipette up and down to mix gently the concentrate can be stored at 4 C for 5 days Add 30 ul of Detection Antibody Concentrate into a tube with 570 ul of Blocking Buffer Mix gently to prepare IX Biotinylated Anti phosphotyrosine Alexa Fluor 555 Conjugated Streptavidin briefly spin the Alexa Fluor555 Conjugated Streptavidin before use Add 50 ul of Blocking Buffer into the tube to prepare a Streptavidin Concentrate Pipette up and down to mix gently Add 10 ul of Streptavidin Concentrate into a tube with ml of Blocking Buffer Mix gently to prepare 1X Streptavidin solution 6 RayBio Human RTK Phosphorylation Antibody Array G series 1 Protocol V Overview and General Considerations A Preparation of Samples The cell can be prepared using following conventional way For attached cells remove supernatant from cell culture wash cells twice with cold IX PBS for suspension cells pellet the cells by spinning
16. vated receptors After incubation with Alexa Fluor 555 Conjugated Streptavidin the signals were visualized by laser scanner using the cy3 channel 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 target protein receptors Bands were visualized with Streptavidin HRP followed by chemiluminescent detection substrate RayBio Human RTK Phosphorylation Antibody Array G series NEG NEG NEG NEG NEG NEG ALK ALK ALK Axl Axl Axl Blk Blk EphA1 EphA1 EphA1 EphA2 EphA2 EphA2 EphA3 EphA3 EphA3 EphA4 EphA4 EphA4 EphA5 EphA5 EphB3 EphB3 EphB3 B4 EphB4 EphB4 B4 B4 B4 FAK FAK FR1 FR1 FR1 FR FGFR GFR2 FGFR2 FGFR2 FGFR2 Fgr Fgr Fgr FRK FRK FRK Fyn Fyn Fyn Hck Hck Hck a isoform a isoform a isoform Eph EphB6 EphB6 EphB6 ErbB2 ErbB2 ErbB2 ErbB3 ErbB3 ON DG BEN 10 JAK2 JAK2 JAK2 JAK3 JAK3 JAK3 LCK LCK LCK LTK LTK LTK Lyn Lyn Lyn 12 PDGFR B PDGFR B PDGFR B PYK2 PYK2 PYK2 RET RET RET ROR1 ROR1 ROR1 ROR2 ROR2 ROR2 14 Tec Tec Tec Tie 1 Tie 1 Tie 1 Tie 2 Tie 2 Tie 2 TNK1 TNK1 TNK1 TRKB TRKB TRKB 16 VEGFR3 VEGFR3 VEGFR3 ZAP70 ZAP70 ZAP70 NEG NEG NEG NEG NEG NEG POS4 POS4 POS4 15 RayBio Human RTK Phosphorylation Antibody Array G s
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