Quantitative analysis of two-dimensional electrophoretograms

Abstract

A method for quantitative analysis of complex film density distributions in autoradiograms is described. The method is intended particularly for measuring the distribution of radioactivity among the proteins resolved by two-dimensional gel electrophoresis but should, of course, be suited to analyzing other two dimensional separations. The film density distribution is first digitized by a high speed rotating drum scanner to generate the image data array that is stored on a magnetic disk. Subsequent analysis involves: 1) data averaging, 2) detection of contours and of their locations, 3) splitting of overlapping spots, 4) conversion of film density to radioactive intensity by means of calibration films, and 5) differentiation and integration to measure the total radioactivity contained in the protein which generates a spot in the autoradiogram. The product of the analysis is a numbered contour map and a table listing coordinates and radioactivity content of each resolved spot. Coordinate transformations for comparison and matching of autoradiograms are also described. A set of utility programs print and graph the data at intermediate stages of the analysis in order to facilitate the checking of procedures and programs.