title: Determining absolute protein numbers by quantitative fluorescence microscopy.
abstract: Biological questions are increasingly being addressed using a wide range of quantitative analytical tools to examine protein complex composition. Knowledge of the absolute number of proteins present provides insights into organization, function, and maintenance and is used in mathematical modeling of complex cellular dynamics. In this chapter, we outline and describe three microscopy-based methods for determining absolute protein numbers--fluorescence correlation spectroscopy, stepwise photobleaching, and ratiometric comparison of fluorescence intensity to known standards. In addition, we discuss the various fluorescently labeled proteins that have been used as standards for both stepwise photobleaching and ratiometric comparison analysis. A detailed procedure for determining absolute protein number by ratiometric comparison is outlined in the second half of this chapter. Counting proteins by quantitative microscopy is a relatively simple yet very powerful analytical tool that will increase our understanding of protein complex composition.
authors: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors: Jolien Suzanne
authors: JS
authors: Verdaasdonk
authors: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors: Josh
authors: J
authors: Lawrimore
authors: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors: Kerry
authors: K
authors: Bloom
mesh_terms: D003598:Cytoskeletal Proteins / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods / Q000592:standards; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D018386:Kinetochores / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods / Q000592:standards; D038761:Photobleaching; D012015:Reference Standards; D004718:Saccharomycetales / Q000378:metabolism; D059629:Signal-To-Noise Ratio
chemical_list: D003598:Cytoskeletal Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Counting; FCS; Fluorescence; Fluorescence standards; Photobleaching; Quantitative imaging; Ratiometric