Simple centrifugation method for efficient pelleting of both small and large unilamellar vesicles that allows convenient measurement of protein binding.
Separation of unilamellar model membrane vesicles from external solution is often an important step in quantitation of vesicle bound or entrapped materials. An efficient method that allows pelleting of both small and large model membrane vesicles by centrifugation is described in this report. In this method streptavidin is added to vesicles containing a trace amount of biotinylated lipid. The resulting aggregation allows pelleting of the vesicles using an ordinary high-speed centrifuge. Control experiments show that the addition of streptavidin does not induce substantial vesicle fusion or leakage of substances trapped in the internal aqueous compartment of the vesicles. The method can accommodate different phospholipid compositions and lipid concentrations. Experiments with proteins that switch between hydrophilic and hydrophobic states show that the method can readily be used to monitor protein binding to vesicles.
Tortorella
D
D
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook 11794-5215.
Ulbrandt
N D
ND
London
E
E
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001710:Biotin; D002498:Centrifugation / Q000379:methods*; D015786:Cytochromes b5 / Q000378:metabolism; D004167:Diphtheria Toxin / Q000378:metabolism; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers / Q000302:isolation & purification* / Q000378:metabolism*; D008561:Membrane Fusion; D026901:Membrane Transport Proteins; D008956:Models, Chemical; D010316:Particle Size; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D011485:Protein Binding; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D019809:Streptavidin
D001426:Bacterial Proteins; D004167:Diphtheria Toxin; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D026901:Membrane Transport Proteins; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D000069816:SEC Translocation Channels; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; D001710:Biotin; D019809:Streptavidin; D015786:Cytochromes b5; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins; C017251:1,2-oleoylphosphatidylcholine