A quantitative electrophoretic migration shift assay for analyzing the specific binding of proteins to lipid ligands in vesicles or micelles.
We present a new assay for analyzing the specific binding of proteins to lipid ligands contained within vesicles or micelles. This assay, referred to as the electrophoretic migration shift assay, was developed using a model system composed of cholera toxin and of its physiological receptor, monosialoganglioside GM1. Using polyacrylamide gel electrophoresis in non-denaturing conditions, the migration of toxin components known to interact with GM1 was retarded when GM1 was present in either lipid vesicles or micelles. This effect was specific, as the migration of proteins not interacting with GM1 was not modified. The localization of retarded proteins and of lipids on gels was further determined by autoradiography. The stoichiometry of binding between cholera toxin and GM1 was determined, giving a value of five GM1 per one pentameric assembly of cholera toxin B-subunits, in agreement with previous studies. The general applicability of this assay was further established using both streptavidin and annexin V together with specific lipid ligands. This assay is fast, simple, quantitative, and requires only microgram quantities of protein.
Arnold
M
M
Laboratoire de Génétique Moléculaire des Eucaryotes, CNRS, Strasbourg, France.
Ringler
P
P
Brisson
A
A
D017304:Annexin A5 / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D002772:Cholera Toxin / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005677:G(M1) Ganglioside / Q000737:chemistry; D066298:In Vitro Techniques; D008024:Ligands; D008081:Liposomes; D008563:Membrane Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D010713:Phosphatidylcholines / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D019809:Streptavidin
D017304:Annexin A5; D001426:Bacterial Proteins; D008024:Ligands; D008081:Liposomes; D008563:Membrane Lipids; D008565:Membrane Proteins; D008823:Micelles; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D005677:G(M1) Ganglioside; C042511:1,2-dioleoylphosphatidylserine; D002772:Cholera Toxin; D019809:Streptavidin; C017251:1,2-oleoylphosphatidylcholine