Quantitation of Cellular Proteins by Flow Cytometry

Quantitation of specific proteins in complex mixtures is simplified by the use of antibodies directed against the protein of interest. If the specific protein is differentially expressed within a population of cells, quantitation of the protein in cell lysates by immunoblotting will provide an average quantity of the protein per cell. As a result, when lysates of different cell populations are compared, large changes in the amount of a protein in a small percentage of cells cannot be distinguished from small changes in the amount of a protein in a large percentage of cells. Flow cytometric analysis solves this problem by providing a means to measure the amount of a specific protein within each individual cell in a population. A fluorescently labeled probe, usually an antibody, is required for detection of the protein. A single cell suspension is reacted with an antibody and passed through a flow cytometer, which focuses the cell suspension into a stream that intersects a laser beam. As each cell passes through the beam, the fluorescent probe in each cell is excited and photomultiplier tubes register the degree of fluorescence (1). Multivariate analysis, the simultaneous measurement of multiple parameters, is a powerful feature of flow cytometry. When determining the quantity of a specific protein in a cell, the quantity of other molecules in the same cell can also be measured by using additional fluors specifically targeted to other molecules. Examples of second molecules measured simultaneously include DNA (2–4) or additional proteins (5). In addition, measurement of light scatter can provide information about cell size and granularity. Most flow cytometers are capable of measuring six colors plus light scatter, but specialized flow cytometers that are capable of measuring as many as eighteen colors are now available.