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Protein microarray assay for the screening of SH3 domain interactions

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Abstract

Analysis of cellular signal transduction processes increasingly focuses on the systematic characterization of complete protein interaction networks. Understanding the interplay of signaling components enables insight into the molecular basis of diverse diseases such as cancer. This paves the way for the rational design of specific therapeutics. Protein interactions are often mediated by conserved modular domains, e.g., SH3-domains, which recognize proline-rich sequences in their cognate ligands. In the course of this study, different microarray formats (reactive silane monolayers and nitrocellulose on glass slides) and assay work flows were evaluated to develop a microarray based screening assay that permits the reliable identification of interactions between certain target proteins with a set of SH3 domains. Nine representative SH3 domains which were produced and purified as GST-fusion proteins were spotted on the microarray substrates and probed with two well-characterized ligands, the Nef protein from HIV-1 and the human protein Sam68. The best results from these low-density model arrays were obtained with nitrocellulose slides. We show that a straightforward and highly robust detection of ligand binding is achieved by staining with a fluorescently labeled antibody directed against the N-terminal His-tag attached to these proteins. The optimized assay protocol reported here allows for the identification of SH3-interactions with high reproducibility and adequate signal-to-background and signal-to-noise ratios, as well as the quantitative determination of relative binding affinities.

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Abbreviations

APTES:

(3-Aminopropyl-)triethoxysilane here simplified as “aminosilane”

CFP:

Cyan fluorescent protein

EVH1:

Ena/VASP homology 1

FITC:

Fluorescein isothiocyanate

FRET:

Förster resonance energy transfer

GPTS:

(3-Glycidyloxypropyl)trimethoxy-silane here simplified as “epoxysilane”

GST:

Glutathione-S-transferase

HRP:

Horseradish peroxidase

MHS:

6-Maleinimidohexanoic acid-N-hydroxysuccinimide ester

Nef:

Negative factor (the term is a misnomer)

Ni-NTA-ATTO647N:

Atto 647 N-Ni2+-nitrilotriacetic acid conjugate

PBS:

Phosphate-buffered saline

PDZ:

Postsynaptic density/disk large/ZO1

PH:

Pleckstrin homology

RasGAP:

Ras GTPase activating protein

S/B:

Signal-to-background

S/N:

Signal-to-noise

Sam68:

Src-associated in mitosis of 68 kDa

SH2/SH3:

Src-homology 2/3 domain

Src:

Sarcoma

WW:

Domain with two conserved tryptophanes

YFP:

Yellow fluorescent protein

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Acknowledgments

We thank Barbara Goricnik for spotting and evaluation of the nitrocellulose slides, Prof. Bo Liedberg (Division of Molecular Physics, Department of Physics, Chemistry and Biology, Linköping University, Sweden) for the ellipsometric measurements

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Authors and Affiliations

  1. Institute of Medical Microbiology and Hygiene, University of Regensburg, Franz-Josef-Strauß-Allee 11, 93053, Regensburg, Germany

    Benedikt Asbach & Ralf Wagner

  2. Geneart AG, BioPark, Josef-Engert-Str. 11, 93053, Regensburg, Germany

    Michael Liss & Ralf Wagner

  3. Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany

    Michaela Kolb & Michael Schäferling

Authors
  1. Benedikt Asbach
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  2. Michaela Kolb
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  3. Michael Liss
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  4. Ralf Wagner
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  5. Michael Schäferling
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Corresponding author

Correspondence to Michael Schäferling.

Additional information

This article was published in the special issue Optical Biochemical and Chemical Sensors (Europtrode X) with Guest Editor Jiri Homola.

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Asbach, B., Kolb, M., Liss, M. et al. Protein microarray assay for the screening of SH3 domain interactions. Anal Bioanal Chem 398, 1937–1946 (2010). https://doi.org/10.1007/s00216-010-4202-x

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  • DOI: https://doi.org/10.1007/s00216-010-4202-x

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