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

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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

Figure 27.4.1 Schematic diagram of overall protocol. Yeast clones containing cDNA constructs are plated on selection medium. Each clone is grown and protein expression is induced. Pellets of each clone are transferred into 96‐well boxes. Fusion proteins are purified and transferred to 96‐well titer plates for printing on slides. In the upper right, the kinase‐treated block shows spots, which indicate phosphorylation of printed proteins with radioactive phosphate. Spots in the corner rectangles are positive controls.

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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
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