Chapter 9: Counting proteins in living cells by quantitative fluorescence microscopy with internal standards.
This chapter describes how a confocal microscope can be treated as a spectrophotometer to measure the absolute number of fluorescent molecules in live cells (Wu and Pollard, 2005).(1) The method provides dynamic range of over three orders of magnitude for counting the number of molecules in a single cell. We present a step-by-step guide to measure concentrations in vivo, explaining many of the practical considerations for using this technique. This chapter is meant as a resource for cell biologists, biochemists, and biophysicists interested in quantifying macromolecules involved in their favorite molecular pathways in live cells.
Wu
Jian-Qiu
JQ
Department of Molecular Genetics , The Ohio State University, Columbus, Ohio 43210, USA.
McCormick
Chad D
CD
Pollard
Thomas D
TD
D002477:Cells / Q000737:chemistry / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000032:analysis; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012568:Schizosaccharomyces / Q000737:chemistry / Q000648:ultrastructure
D011506:Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins