Functional Assays on High‐Content Protein Microarrays

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 [2,4,6,7,17,17‐³ H(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‐³³ P]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‐³ H]adenosyl‐L‐methionine ([³ H]SAM; Perkin Elmer, cat. no. NET155V250UC)
[2,4,6,7,17,17‐³ H(N)]estradiol, ([³ H]estradiol; Perkin Elmer, cat. no. NET517250UC)
Methyltransferase assay buffer (see recipe )
100 mM NaHCO₃ , 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

Figure 1. Anti‐GST image of over 9000 unique human proteins on a ProtoArray human protein microarray. One of the subarrays is enlarged to show the subarray layout. Multiple control proteins are shown. These control proteins help to orient the array grid in the data analysis step and serve as positive and negative controls for different applications.

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Figure 2. Schematic of the ubiquitin ligase profiling workflow. A typical assay involves probing five arrays. Three of the arrays are probed with E3 ligases at three different concentrations. The other two arrays are negative controls which are probed with biotin‐ubiquitin only or biotin‐ubiquitin plus E1 and E2 ligases.

View Image

Figure 3. CMK1 protein‐protein interaction assay. A ProtoArray image probed with V5‐CMK1 shows the interaction of calmodulin with V5‐CMK1. The positive control, V5 control protein, is also detected by the anti‐V5 antibody. *The calmodulin signals did not show up on the negative array (data not shown).

View Image

Figure 4. MAPK14/ p38γ kinase substrate identification assay. The ATP‐only treatment of the ProtoArray shows the autophosphorylation of many printed kinases. In comparison, the MAPK14/p38γ–reated array shows additional phosphorylation spots, such as the inactive MAPKAP kinase in the enlarged region.

View Image

Figure 5. PRMT1 methyltransferase profiling assay. The image of the ProtoArray treated with PRMT1 shows some methylation spots, such as SERBP1 and C1orf77 in the enlarged region. Both proteins are known substrates of PRMT1. In this array image, ERα spots also show up since the array was also treated with ³ H[estradiol]. The ERα spots are landmarks for data acquisition.

View Image

Videos

Literature Cited

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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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Functional Assays on High‐Content Protein Microarrays

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited