Differential binding studies applying functional protein microarrays and surface plasmon resonance.
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.
Seitz
Harald
H
Max-Planck Institute for Molecular Genetics, Department of Vertebrate Genomics, Berlin, Germany. seitz@molgen.mpg.de
Hutschenreiter
Silke
S
Hultschig
Claus
C
Zeilinger
Carsten
C
Zimmermann
Bastian
B
Kleinjung
Frank
F
Schuchhardt
Johannes
J
Eickhoff
Holger
H
Herberg
Friedrich W
FW
D015374:Biosensing Techniques; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D009414:Nerve Growth Factors / Q000737:chemistry / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D040081:Protein Array Analysis; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D000075383:S100 Calcium Binding Protein A6; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance
D018797:Cell Cycle Proteins; D009414:Nerve Growth Factors; D009842:Oligopeptides; D011994:Recombinant Proteins; D000075383:S100 Calcium Binding Protein A6; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579160:S100B protein, human; C097755:arginyl-glycyl-serine; C055351:S100A6 protein, human