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J Vis Exp. 2014 Nov 29;(93):e51937. doi: 10.3791/51937.

Real time measurements of membrane protein:receptor interactions using Surface Plasmon Resonance (SPR).

Author information

1
Department of Biochemistry, The Bruce and Ruth Rappaport Faculty of Medicine, The Technion-Israel Institute of Technology.
2
Department of Biochemistry, The Bruce and Ruth Rappaport Faculty of Medicine, The Technion-Israel Institute of Technology; lewinson@tx.technion.ac.il.

Abstract

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

PMID:
25489923
PMCID:
PMC4354417
DOI:
10.3791/51937
[Indexed for MEDLINE]
Free PMC Article

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