Preparation of Recombinant Protein Spotted Arrays for Proteome‐Wide Identification of Kinase Targets

Hogune Im1, Michael Snyder1

1 Department of Genetics, Stanford University, Stanford, California
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 27.4
DOI:  10.1002/0471140864.ps2704s72
Online Posting Date:  April, 2013
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Abstract

Protein microarrays allow unique approaches for interrogating global protein interaction networks. Protein arrays can be divided into two categories: antibody arrays and functional protein arrays. Antibody arrays consist of various antibodies and are appropriate for profiling protein abundance and modifications. Functional full‐length protein arrays employ full‐length proteins with various post‐translational modifications. A key advantage of the latter is rapid parallel processing of large number of proteins for studying highly controlled biochemical activities, protein‐protein interactions, protein‐nucleic acid interactions, and protein‐small molecule interactions. This unit presents a protocol for constructing functional yeast protein microarrays for global kinase substrate identification. This approach enables the rapid determination of protein interaction networks in yeast on a proteome‐wide level. The same methodology can be readily applied to higher eukaryotic systems with careful consideration of overexpression strategy. Curr. Protoc. Protein Sci. 72:27.4.1‐27.4.14. © 2013 by John Wiley & Sons, Inc.

Keywords: protein array; post‐translation; phosphorylation; kinase

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Protein Induction and Purification of Proteins for Printing
  • Basic Protocol 2: Printing of Proteome Microarrays
  • Basic Protocol 3: Perform Radioactive In Vitro Kinase Assay on a Protein Microarray
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Protein Induction and Purification of Proteins for Printing

  Materials
  • TAP‐tagged yeast strains (see Strategic Planning)
  • SC‐ura medium, agar plates and liquid (see recipe)
  • YP + 6% galactose (see recipe)
  • Milli‐Q water, ice‐cold
  • Lysis buffers 1, 2, and 3 (see reciperecipes)
  • Elution buffer (see recipe)
  • IgG Sepharose 6 Fast Flow (GE Healthcare Life Sciences, cat. no. 17‐0969‐02)
  • Paint shaker (Harbil 5G‐HD) or bead beater (MP bioscience)
  • GST‐3C (prepared in‐house) or PreScission 3C protease (GE Healthcare Life Sciences, cat. no. 27‐0843‐01)
  • Histone H1 (Sigma)
  • 96‐pin replicator device (Boekel, cat. no. 140500)
  • 30°C incubator with shakers fitted for 50‐ml conical tubes
  • 96‐well deep‐well round‐bottom plate (Nunc)
  • 3.5‐mm glass beads, autoclaved (PGC Scientifics, cat. no. 41‐5500‐06)
  • Multichannel pipettor: 2‐ to 20‐µl, 5‐ to 100‐µl, and 1‐ml
  • Gas‐permeable seal (Qiagen, Airpore)
  • 50‐ml flip‐top conical tubes
  • 8‐mm glass beads, autoclaved (PGC Scientifics, cat. no. 41‐5500‐21)
  • Tabletop centrifuge with 50‐ml conical tube and microplate carriers (Sorvall, cat. no. RTH‐250)
  • Vortex mixer
  • 96‐well deep‐well plates with fitted silicone mat seals (Dot Scientific, cat. no. R6530)
  • Paper towels
  • 0.5‐mm glass beads (USA Scientific, cat. no. 7400‐2405)
  • Platform rotator
  • Wide‐bore pipet tips, 200 µl
  • 1.2‐µm pore hydrophilic PVDF filter plates (Millipore)
  • 96‐well deep‐well round‐bottom plates with fitted silicone mat seals (Dot Scientific, cat. nos. PC92271‐NS9, R618‐NS9)
  • 96‐well microtiter plates (USA Scientific, cat. nos. 1830‐9610)
  • 0.65‐µm pore hydrophilic low‐protein‐binding filter plates (Millipore)
  • Glutathione Sepharose beads (e.g., Glutathione Sepharose 4B; GE Healthcare Life Sciences, cat. no. 27‐4574‐01)
  • 384‐well flat‐bottom polypropylene plates

Basic Protocol 2: Printing of Proteome Microarrays

  Materials
  • 30% glycerol
  • Pin/pin head cleaning solution (Arrayit), optional
  • 384‐well plate containing purified protein samples ( protocol 1)
  • 48‐pin contact array printer (Genomics Solutions)
  • 384‐well plate
  • Kimwipes
  • UltraGAPS slides (Corning Life Sciences)
  • Glass slides
  • Centrifuge

Basic Protocol 3: Perform Radioactive In Vitro Kinase Assay on a Protein Microarray

  Materials
  • Three protein microarrays per kinase to be probed (e.g., from protocol 2)
  • Superblock/0.1% Triton X‐100 (Thermo/Fisher)
  • Kinase to be assayed, freshly prepared (1 to 50 nM is required per probing)
  • Kinase buffer (see recipe)
  • Wash buffer (see recipe)
  • Milli‐Q water
  • 1‐ml syringes
  • 0.45‐µm filter tips
  • Hybrislip hybridization coverslips (Grace Bio Labs)
  • Humidified chamber
  • 50‐ml conical tubes
  • Tabletop centrifuge
  • Plastic wrap
  • Autoradiography film
  • High‐resolution scanner
  • Photoshop
  • GenePix Software
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Figures

Videos

Literature Cited

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Internet Resources
  http://www.invitrogen.com/site/us/en/home/LINNEA‐Online‐Guides/LINNEA‐Guide‐to‐Clones/Ultimate‐ORF‐Clones.html
  Invitrogen's Ultimate ORF clone information Web site has more details on available cDNA collections and expression strategy using GATEWAY technology
  http://www.invitrogen.com/site/us/en/home/Products‐and‐Services/Applications/Protein‐Expression‐and‐Analysis/Biomarker‐Discovery/ProtoArray/Resources/Data‐Analysis.html
  Information about Invitrogen's Prospector software to analyze protein array data.
  http://www.openbiosystems.com
  Web site for OpenBiosystems, a distributor of many of cDNA collection now owned by Thermo.
  http://www.origene.com
  Web site for Origene. It houses expression validated cDNA clones.
  http://dnasu.asu.edu/DNASU
  Web site for the ASU biodesign institute. An academic resource for obtaining various mammalian cDNA collections and expression vector plasmid for nominal fee.
  http://ymd.med.yale.edu/kei‐cgi/kc_mac_dev8.pl
  MicroArray Convolutor. Web tool for generating gal file for microarray analysis.
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