Ultraviolet imaging densitometry of unstained gels for two-dimensional electrophoresis

A system suitable for ultraviolet imaging densitometry of two-dimensional electrophoretic gels that are unstained is described, together with its applications. A flying-spot densitometer linked with a personal computer was used for data acquisition, generation of mapping data, and image processing. Randomly distributed zones of proteins on two-dimensional gels were detected at 280 nm without being stained by two-dimensional scanning, and the densitometric value of each pixel (0.2 x 0.2 mm) was memorized by the computer, which generated a mapping pattern with density contours. The amount and densitometric value of cytochrome c had a linear relationship in the range of 2-200 micrograms. Zone locations of bovine liver proteins separated on two-dimensional gels were indicated on a map expressed in X-Y coordinates, and the pIs and molecular weights could be calculated from the map by use of pI and molecular weight markers on the same gel.