Quantitation of Cellular Proteins by Laser Scanning Cytometry

Abstract

Flow Cytometry (FC) is a popular method for the simultaneous quantitation of multiple specific proteins and other molecules in each cell of a single cell suspension (1). Laser Scanning Cytometry (LSC) is a closely related technology that provides many of the benefits of FC and offers additional features that allow morphological and biochemical analysis of a cell population (2, 3). In both methods, quantitation of endogenous cellular proteins generally requires antibodies against the protein of interest. These antibodies must be either directly labeled with a fluorochrome or detected by a fluorescently-labeled secondary antibody. Using fluorescently-labeled antibodies in combination with fluorescent dyes that quantitatively bind to DNA, both FC and LSC have been valuable in measuring the quantities of specific proteins in relation to cell cycle position (4). In contrast to FC, where a single cell suspension is passed through a fixed-position laser beam, LSC analyzes cells fixed to a surface such as a microscope slide or cover slip. Since the position of the cell on the slide is part of the recorded data, it is possible to visualize the cell after the initial recording event or to subsequently stain the cell with another fluorescent probe and make a second measurement. LSC also allows analysis of cells following biochemical treatments that are incompatible with analysis by FC. In this chapter we describe the value of LSC in analyzing chromatin-associated proteins. Specifically, we describe an in situ fractionation procedure that allows measurement of total and chromatin-associated MCM2, a DNA helicase subunit, as a function of cell cycle position.