Localization and mobility of bacterial proteins by confocal microscopy and fluorescence recovery after photobleaching.
This chapter describes the use of laser-scanning confocal fluorescence microscopy for determining the localization of fluorescently tagged proteins within bacterial cells, discussing the problems caused by the limited resolution of an optical microscope. It also explains a relatively simple method for using fluorescence recovery after photobleaching (FRAP) to observe and quantify the diffusion of fluorescently tagged proteins in bacterial cells. The techniques are illustrated with reference to measurements on green fluorescent protein (GFP)-tagged proteins in Escherichia coli.
Mullineaux
Conrad W
CW
School of Biological and Chemical Sciences, Queen Mary, University of London, London, UK.
D001426:Bacterial Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004058:Diffusion; D029968:Escherichia coli Proteins / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods*; D019897:Periplasm / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins