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Optical sensor techniques have recently been developed, which allow for the investigation of biomolecular interaction in real time. These techniques rely on the detection of changes in refractive index as ligate binds to a ligand, which is immobilized on a sensor surface. Examples of optical sensor techniques include surface plasmon resonance (3,4) and the resonant mirror (1,2). The application of these systems for the investigation of protein protein interactions necessitates the immobilization of one of the binding partners to the sensor surface. During this procedure, the binding activity of the protein should be retained. However, the latter could be reduced to a certain extent if the ligand is immobilized directly because immobilization can block the binding site or induce conformational changes of the protein. To overcome this problem, indirect immobilization strategies have been used, i.e., avidin covalently bound to the sensor matrix was used to capture biotin-labeled ligands (1). Here we describe an efficient method for the investigation of a protein ligate binding to a recombinant glutathione S-transferase (GST) fusion protein using the resonant mirror technique. Since the direct immobilization of the GST-fusion protein phosphoinositide 3-kinase γ (GST-PI3Kγ) to a carboxymethyl dextran (CMD) matrix resulted in an almost complete loss of its binding activity to added ligate Ras, we developed a novel indirect immobilization procedure. We show that glutathione (GSH), only when immobilized by a 6-carbon spacer, captures GST-PI3Kγ efficiently and retains its binding activity. Under these conditions, specific binding of Ras to GSTPI3Kγ could be observed. The study was performed on the interaction analysis system, IAsys,

Real-Time Assay of the Interaction of a GST Fusion Protein with a Protein Ligate Using Resonant Mirror Technique