SPR-based immunocapture approach to creating an interfacial sensing architecture: mapping of the MRS18-2 binding site on retinoblastoma protein

Biosensor technologies based on optical readout are widely used in protein–protein interaction studies. Here we describe a fast and simple approach to the creation of oriented interfacial architectures for surface plasmon resonance (SPR) transducers, based on conventional biochemical procedures and...

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Bibliographic Details
Published in:Analytical and bioanalytical chemistry 2006-12, Vol.386 (7-8), p.2063-2073
Main Authors: Snopok, Boris, Yurchenko, Mariya, Szekely, Laszlo, Klein, George, Kashuba, Elena
Format: Article
Language:English
Subjects:
antibodies
Antibodies - immunology
binding sites
Binding Sites - immunology
biosensors
Cell Line, Tumor
Glutathione Transferase - genetics
Glutathione Transferase - metabolism
guanidines
Herpesvirus 4, Human
Humans
Immunoassay - methods
Protein Binding
protein-protein interactions
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Retinoblastoma Protein - chemistry
Retinoblastoma Protein - immunology
Retinoblastoma Protein - metabolism
surface plasmon resonance
Surface Plasmon Resonance - methods
tumor suppressor proteins
two hybrid system techniques
Viral Proteins - metabolism
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Summary:Biosensor technologies based on optical readout are widely used in protein–protein interaction studies. Here we describe a fast and simple approach to the creation of oriented interfacial architectures for surface plasmon resonance (SPR) transducers, based on conventional biochemical procedures and custom reagents. The proposed protocol permits the oriented affinity-capture of GST fusion proteins by a specific antibody which is bound to protein A, which in turn has been immobilized on the transducer surface (after the surface has been modified by guanidine thiocyanate). The applicability of the method was demonstrated by studying the interaction between retinoblastoma tumor suppressor protein (pRb) and MRS18-2 proteins. The formation of the pRb–MRS18-2 protein complex was examined and the pRb binding site (A-box–spacer–B-box) was mapped. We have also shown that MRS18-2, which was detected as the Epstein–Barr virus-encoded EBNA-6 binding partner using the yeast two-hybrid system, binds to pRb in GST pull-down assays.
ISSN:1618-2642
1618-2650
DOI:10.1007/s00216-006-0867-6