title: A cell-free assay to determine the stoichiometry of plasma membrane proteins.
abstract: Plasma membrane receptors, transporters, and ion channel molecules are often found as oligomeric structures that participate in signaling cascades essential for cell survival. Different states of protein oligomerization may play a role in functional control and allosteric regulation. Stochastic GFP-photobleaching (SGP) has emerged as an affordable and simple method to determine the stoichiometry of proteins at the plasma membrane. This non-invasive optical approach can be useful for total internal reflection of fluorescence microscopy (TIRFM), where signal-to-noise ratio is very high at the plasma membrane. Here, we report an alternative methodology implemented on a standard laser scanning confocal microscope (LSCM). The simplicity of our method will allow for its implementation in any epifluorescence microscope of choice.
authors.affiliation: Instituto de Fisiología, Facultad de Medicina, Universidad Austral de Chile, Campus Isla Teja, Valdivia, Chile.
authors.forename: Cesar
authors.initials: C
authors.lastname: Trigo
authors.forename: Juan P
authors.initials: JP
authors.lastname: Vivar
authors.forename: Carlos B
authors.initials: CB
authors.lastname: Gonzalez
authors.forename: Sebastian
authors.initials: S
authors.lastname: Brauchi
mesh_terms: D002462:Cell Membrane / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000032:analysis*; D057809:HEK293 Cells; D006801:Humans; D007473:Ion Channels / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis*; D018613:Microscopy, Confocal / Q000379:methods*; D038761:Photobleaching
chemical_list: C510235:HVCN1 protein, human; D007473:Ion Channels; D008565:Membrane Proteins; D049452:Green Fluorescent Proteins