Apparatus for direct counting of beta-rays from two-dimensional protein gels: measurement of changes in protein synthesis due to changes in density of Chinese hamster ovary cells.

Abstract

A method is described for scanning twodimensional protein gels that utilizes direct counting of a-rays instead of autoradiography. The methodology is compared with autoradiographic results and data are presented demonstrating changed patterns of protein synthesis accompanying changes in cell density. The method is rapid and permits identification of differences in protein abundance of -10% for a substantial fraction of the more prominent proteins. A modulation effect of >5 standard deviations is shown to occur for an appreciable number of the proteins that accompany the inhibition of cell growth due to contact inhibition. The method promises application to a variety of biochemical and genetic problems designed to delineate changes in protein synthesis accompanying changes in genome, molecular environment, history, and state of differentiation of the cell populations studied.The combination of somatic cell genetics and recombinant DNA methodologies has made possible an enormous increase in the elucidation of the structure of the mammalian cell genome. However, perhaps the central unsolved problem of human genetics revolves around the following question: How are the cells of the different tissues, despite their possession of identical chromosomal and DNA structures, able to produce different and specific patterns of protein biosynthesis? Understanding of this process will illuminate the regulatory mechanisms that govern gene expression and the molecular dynamics of disease in the various tissues. Therefore, it becomes necessary to be able to study the spectrum of protein biosynthesis of different cell populations and to correlate changes in this pattern with changes in the DNA state, the molecular environment, and the differentiation history of the cell, with precision.The two-dimensional (2D) gel technique pioneered by O'Farrell (1) offers great power in elucidating protein biosynthetic patterns since it permits examination of large numbers of the proteins synthesized by any cell population. The present study was undertaken to eliminate the necessity of employing autoradiography to visualize the points of deposition in the gel of the peptides containing radioactively labeled amino acids and to measure changes in quantity with precision. An exposure of autoradiographic films can be time consuming and a linear relationship between the amount of film blackening and quantity of peptide deposited is obtained only over a relatively limited range of intensities, making quantitative comparisons difficult. Quantification using autoradiography requires a series of autoradiographs with different exposure times (2).We describe here a method for detection and quantitation of radiolabeled peptides in 2D gels, in which the a-rays are counted directly. The methodology is described, its results are compared with the autoradiographic technique, and some representative biological findings are reported. In this study we have concentrated attention on the group of the most abundant proteins synthesized by the Chinese hamster ...

Photoaffinity labeling of Escherichia coli RNA polymerase/poly[d(A-T)] transcription complexes by nascent RNA

“…The 3-mm width of the gel slices is too large to take full advantage of the resolution afforded by electrophoresis but narrow enough to produce hundreds of slices to be counted. The slicing procedure appears to be the limiting factor in our current methodology; automated methods would be expected to produce less scatter in the final results (Anderson et al, 1987).…”