Differential binding studies applying functional protein microarrays and surface plasmon resonance.

Harald Seitz; Silke Hutschenreiter; Claus Hultschig; Carsten Zeilinger; Bastian Zimmermann; Frank Kleinjung; Johannes Schuchhardt; Holger Eickhoff; Friedrich W Herberg

doi:10.1002/pmic.200500181

PubMed · 16912968

Differential binding studies applying functional protein microarrays and surface plasmon resonance.

Harald Seitz·Silke Hutschenreiter·Claus Hultschig·Carsten Zeilinger·Bastian Zimmermann·Frank Kleinjung·Johannes Schuchhardt·Holger Eickhoff·Friedrich W Herberg

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Abstract

A variety of different in vivo and in vitro technologies provide comprehensive insights in protein-protein interaction networks. Here we demonstrate a novel approach to analyze, verify and quantify putative interactions between two members of the S100 protein family and 80 recombinant proteins derived from a proteome-wide protein expression library. Surface plasmon resonance (SPR) using Biacore technology and functional protein microarrays were used as two independent methods to study protein-protein interactions. With this combined approach we were able to detect nine calcium-dependent interactions between Arg-Gly-Ser-(RGS)-His6 tagged proteins derived from the library and GST-tagged S100B and S100A6, respectively. For the protein microarray affinity-purified proteins from the expression library were spotted onto modified glass slides and probed with the S100 proteins. SPR experiments were performed in the same setup and in a vice-versa approach reversing analytes and ligands to determine distinct association and dissociation patterns of each positive interaction. Besides already known interaction partners, several novel binders were found independently with both detection methods, albeit analogous immobilization strategies had to be applied in both assays.

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Biosensing Techniques Cell Cycle Proteins Humans Nerve Growth Factors Oligopeptides Protein Array Analysis Protein Binding Protein Interaction Mapping Recombinant Proteins S100 Calcium Binding Protein A6 S100 Calcium Binding Protein beta Subunit S100 Proteins Surface Plasmon Resonance Comparative Study Journal Article Research Support, Non-U.S. Gov't

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Harald Seitz, Silke Hutschenreiter, Claus Hultschig, Carsten Zeilinger, Bastian Zimmermann, Frank Kleinjung, Johannes Schuchhardt, Holger Eickhoff, Friedrich W Herberg. 2006. Differential binding studies applying functional protein microarrays and surface plasmon resonance.. https://doi.org/10.1002/pmic.200500181

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