Functional Assays on High‐Content Protein Microarrays

Lihao Meng1

1 Life Technologies, Carlsbad, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch110267
Online Posting Date:  September, 2012
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Abstract

High‐content functional protein microarrays, such as ProtoArray Human Protein Array from Life Technologies, contain thousands of proteins or protein fragments of biological significance. Based on this platform, numerous assays have been developed to investigate protein‐protein interactions, protein‐DNA interactions, protein‐phospholipid interactions, small‐molecule targets, substrates of protein kinases, substrates of ubiquitin E3 ligases, and substrates of methyltransferase. This article provides a detailed illustration of the general procedure for setting up functional assays for high‐throughput screening and also provides guidelines for data analysis. The protocols include the use of fluorescently labeled and radioactive probes for detection. Curr. Protoc. Chem. Biol. 4:211‐231 © 2012 by John Wiley & Sons, Inc.

Keywords: high content protein microarray; functional assays; protein‐protein interaction; small‐molecule interaction; kinase substrate profiling; ubiquitin ligase substrate profiling; protein methyltransferase substrate profiling

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Protein‐Protein Interaction (PPI) Assay on the ProtoArray Human Protein Array
  • Basic Protocol 2: Small‐Molecule Interaction (SMI Radioactive) Assay on the ProtoArray Human Protein Array
  • Basic Protocol 3: Kinase Substrate Identification (KSI) Assay on the ProtoArray Human Protein Array
  • Basic Protocol 4: Ubiquitin/SUMO Ligase Substrate Profiling Assay on the ProtoArray Human Protein Array
  • Basic Protocol 5: Methyltransferase Profiling Assay on the ProtoArray Human Protein Array
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Protein‐Protein Interaction (PPI) Assay on the ProtoArray Human Protein Array

  Materials
  • ProtoArray blocking buffer (see recipe)
  • ProtoArray washing buffer (see recipe)
  • Array control protein: 6 µg per slide (BioEase V5‐labeled CMK1, Invitrogen, cat. no.476064)
  • Alexa Fluor 647 anti‐V5 antibody (Molecular Probes, custom)
  • ProtoArray human protein microarray slides v5.0 (Invitrogen, cat. no. 260044)
  • 4‐well trays (quadri‐PERM ‐4‐chamber culture dish, ISC Bioexpress, cat. no. T‐2896‐1, http://www.bioexpress.com/)
  • Lab rotator (Lab‐Line Instruments, cat. no. 1314)
  • Forceps
  • LifterSlip coverslips (Erie Scientific, cat. no. 25x601‐2‐4789, http://www.thermofisher.com/)
  • Polyacetal slide rack (RA Lamb, cat. no. E99, http://www.thermofisher.com/)
  • 50‐ml conical tubes (VWR, cat. no. 89004‐367), sterile
  • Eppendorf centrifuge (5810) (Fisher Scientific, cat. no. 05‐400‐60) with microtiter plate adapter for rotor
  • GenePix 4000B microarray scanner (Molecular Devices)
  • GenePix Pro software (Molecular Devices)
  • ProtoArray Prospector v.5 (available at http://www.invitrogen.com/site/us/en/home/Products‐and‐Services/Applications/Protein‐Expression‐and‐Analysis/Biomarker‐Discovery/ProtoArray/Online‐Tools.htmlas a free download)

Basic Protocol 2: Small‐Molecule Interaction (SMI Radioactive) Assay on the ProtoArray Human Protein Array

  Materials
  • SMI buffer (see recipe)
  • 40 pCi/µl [2,4,6,7,17,17‐3H(N)]estradiol (Perkin Elmer, cat. no. NET517250UC)
  • ProtoArray human protein microarray slides v5.0 (Invitrogen, cat. no. 260044)
  • 4‐well trays (quadri‐PERM ‐4‐chamber culture dish, ISC Bioexpress, cat. no. T‐2896‐1, http://www.bioexpress.com/)
  • 50‐ml conical tubes (VWR, cat. no. 89004‐367), sterile
  • Forceps
  • LifterSlip coverslips (Erie Scientific, cat. no. 25x601‐2‐4789, http://www.thermofisher.com/)
  • Polyacetal slide rack (RA Lamb, cat. no. E99, http://www.thermofisher.com/)
  • Eppendorf centrifuge (5810) (Fisher Scientific, cat. no. 05‐400‐60) with plate adapter for rotor
  • Transparent adhesive tape
  • Thick filter paper
  • X‐ray film cassette
  • Tritium‐sensitive phosphor imager screen (PerkinElmer, cat. no. 7001489)
  • Cyclone phosphor imager (PerkinElmer)
  • Imaging software (e.g., Adobe Photoshop)
  • GenePix Pro software (Molecular Devices)
  • ProtoArray Prospector v.5 (available at http://www.invitrogen.com/site/us/en/home/Products‐and‐Services/Applications/Protein‐Expression‐and‐Analysis/Biomarker‐Discovery/ProtoArray/Online‐Tools.htmlas a free download)/
NOTE: Array blocking and array probing steps 1 to 4 are carried out at 4°C. All other steps are conducted at room temperature unless stated otherwise.

Basic Protocol 3: Kinase Substrate Identification (KSI) Assay on the ProtoArray Human Protein Array

  Materials
  • Kinase blocking buffer: phosphate‐buffered saline (PBS) containing 1% (w/v) BSA
  • MAPK14/p38γ protein kinase (Invitrogen, cat. no. PV3304)
  • KSI probing buffer (see recipe)
  • 10 mCi/ml [γ‐33P]ATP, sp. act. 3000Ci/mmol (Perkin Elmer, cat. no. NEG302H250UC)
  • Ultrapure 10% SDS Solution (Invitrogen, cat. no. 24730‐020)
  • ProtoArray human or control protein microarray slides v5.0 (Invitrogen, cat. no. 260044/260022)
  • 4‐well trays (quadri‐PERM ‐4‐chamber culture dish, ISC Bioexpress, cat. no. T‐2896‐1, http://www.bioexpress.com/)
  • 50‐ml conical tubes (VWR, cat. no. 89004‐367), sterile
  • Forceps
  • Glass slide coverslips (Sigma, cat. no. H0784‐100EA)
  • 30°C incubator
  • Polyacetal slide rack (RA Lamb, cat. no. E99, http://www.thermofisher.com/)
  • Eppendorf centrifuge (5810) (Fisher Scientific, cat. no. 05‐400‐60) with plate adapter for rotor
  • X‐ray film cassette
  • Clear plastic wrap (e.g., Saran wrap)
  • MS or SR phosphor screen (Perkin Elmer, cat. no. 7001723/7001486)
  • Cyclone phosphor imager (PerkinElmer)
  • Imaging software (e.g., Adobe Photoshop)
  • GenePix Pro software (Molecular Devices)
  • ProtoArray Prospector v.5 (available at http://www.invitrogen.com/site/us/en/home/Products‐and‐Services/Applications/Protein‐Expression‐and‐Analysis/Biomarker‐Discovery/ProtoArray/Online‐Tools.htmlas a free download)

Basic Protocol 4: Ubiquitin/SUMO Ligase Substrate Profiling Assay on the ProtoArray Human Protein Array

  Materials
  • ProtoArray blocking buffer (see recipe)
  • Ubiquitin ligase assay buffer (see recipe)
  • Ubiquitin E1 (BostonBiochem, cat. no. E‐305)
  • Ubiquitin E2 (UbcH5) (BostonBiochem, cat. no. E2‐622)
  • Biotin‐ubiquitin (BostonBiochem, cat. no. U‐570)
  • Energy regenerating solution (BostonBiochem, cat. no. B‐10)
  • Ultrapure 10% SDS Solution (Invitrogen, cat. no. 24730‐020)
  • Streptavidin‐conjugated AlexaFluor 647 (Molecular Probes, cat. no. S‐21374)
  • ProtoArray human protein microarray slides v5.0 (Invitrogen, cat. no. 260044)
  • 4‐well trays (quadri‐PERM ‐4‐chamber culture dish, ISC Bioexpress, cat. no. T‐2896‐1, http://www.bioexpress.com/)
  • Lab rotator (Lab‐Line Instruments, cat. no. 1314)
  • 30°C incubator
  • Forceps
  • LifterSlip coverslips (Erie Scientific, cat. no. 25x601‐2‐4789, http://www.thermofisher.com/)
  • 4‐well trays (quadri‐PERM ‐4‐chamber culture dish, ISC Bioexpress, cat. no. T‐2896‐1, http://www.bioexpress.com/)
  • Polyacetal slide rack (RA Lamb, cat. no. E99, http://www.thermofisher.com/)
  • Eppendorf centrifuge (5810) (Fisher Scientific, cat. no. 05‐400‐60) with plate adapter for rotor
  • GenePix 4000B microarray scanner (Molecular Devices)
  • GenePix Pro software (Molecular Devices)
  • ProtoArray Prospector v.5 (available at http://www.invitrogen.com/site/us/en/home/Products‐and‐Services/Applications/Protein‐Expression‐and‐Analysis/Biomarker‐Discovery/ProtoArray/Online‐Tools.htmlas a free download)

Basic Protocol 5: Methyltransferase Profiling Assay on the ProtoArray Human Protein Array

  Materials
  • ProtoArray blocking buffer (see recipe)
  • Protein arginine methyltransferase (PRMT1; Sigma, cat. no. P0083‐25UG)
  • S‐[methyl‐3H]adenosyl‐L‐methionine ([3H]SAM; Perkin Elmer, cat. no. NET155V250UC)
  • [2,4,6,7,17,17‐3H(N)]estradiol, ([3H]estradiol; Perkin Elmer, cat. no. NET517250UC)
  • Methyltransferase assay buffer (see recipe)
  • 100 mM NaHCO 3, pH 9 (Invitrogen, cat. no. 25080‐094; adjust pH to 9 and dilute 9‐fold to 100 mM; each slide will require 5 ml)
  • TR phosphor screen (Perkin Elmer, cat. no. 7001489)
  • Additional reagents and equipment for blocking array slides ( protocol 1) and radioactive SMI assay ( protocol 2)
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Figures

Videos

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Internet Resources
   http://www.invitrogen.com/site/us/en/home/Products‐and‐Services/Applications/Protein‐Expression‐and‐Analysis/Biomarker‐Discovery/ProtoArray/Human‐Protein‐Microarrays‐Overview.html
  Life Technologies. Overview of ProtoArray Human Protein Microarrays.
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