Real time analysis of intact organelles using surface plasmon resonance.
Membrane proteins remain refractory to standard protein chip analysis. They are typically expressed at low densities in distinct subcellular compartments, their biological activity can depend on assembly into macromolecular complexes in a specific lipid environment. We report here a real-time, label-free method to analyze membrane proteins inserted in isolated native synaptic vesicles. Using surface plasmon resonance-based biomolecular interaction analysis (Biacore), organelle capture from minute quantities of 10,000 g brain supernatant (1-10 microg) was monitored. Immunological and morphological characterization indicated that pure intact synaptic vesicles were immobilized on sensor chips. Vesicle chips were stable for days, allowing repetitive use with multiple analytes. This method provides an efficient way in which to characterize organelle membrane components in their native context. Organelle chips allow a broad range of measurements, including interactions of exogenous ligands with the organelle surface (kinetics, Kd), and protein profiling.
Ferracci
Géraldine
G
Unité de Méthodologie des Interactions Moléculaires, Institut Jean Roche, Faculté de Médecine secteur Nord, 13916 Marseille, France.
Seagar
Michael
M
Joël
Courageot
C
Miquelis
Raymond
R
Lévêque
Christian
C
D000906:Antibodies / Q000276:immunology; D003199:Computer Systems; D007700:Kinetics; D046228:Microarray Analysis; D008854:Microscopy, Electron; D015388:Organelles / Q000276:immunology / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000276:immunology; D012995:Solubility; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*; D013572:Synaptic Vesicles / Q000276:immunology / Q000378:metabolism; D013997:Time Factors
D000906:Antibodies; D011506:Proteins