Radioisotopic Labeling of Proteins for Polyacrylamide Gel Electrophoresis
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The metabolic labeling of proteins with radiolabeled amino acids, either to determine the rate of synthesis or to identify proteins following separation by gel electrophoresis, at first sight appears trivial. However, there are a number of important methodological considerations to be aware of in these procedures. Principal among these is the problem of intracellular amino acid pools and the effects of changing these on the metabolism of the cell. Fortunately, amino acid intracellular pools equilibrate rapidly with the external media, maximally within 5 min for valine and usually within 1 min for amino acids, such as leucine. Thus, providing a large extracellular pool of nonradioactive amino acids is maintained, the radiolabeled essential amino acid is introduced at low concentrations, and labeling continues for at least 10 times the pool equilibration time, accurate protein synthetic rates can be determined. This experimental design, however, can rarely be achieved if labeling of proteins to high specific activity is required, for example, for detection of all but a few of the most abundant cellular proteins following separation by two-dimensional gel electrophoresis. Usually, in these cases, the external amino acid, often methionine and/or cysteine, is removed and substituted with a low concentration of very high specific activity cognate amino acid. This manipulation, however, will inhibit the initiation of ribosomes onto mRNA (1), which, in turn, results in the selective translation of high-efficiency mRNAs (2) and, therefore, the detection of a biased profile of proteins. Furthermore, the reduction in the rate of ribosome initiation will result in a reduced level of radio-active incorporation into proteins. Considerable care, therefore, should be taken in extrapolating the results obtained to physiological circumstances. Thus, as in all experiments, careful thought should be given to the experimental end point. For example, it is often better to reduce the media concentration of the amino acid used to label to a one-tenth or one-hundredth concentration (media amino acids are always available in excess) and label for several hours or to equilibrium, rather than to use short pulses given in the absence of the external amino acid. These conditions, however, would not suffice if the experimenter wishes to analyze proteins synthesized rapidly in response to a changed external stimuli, such as the addition of a growth factor. Bearing these caveats in mind, this chapter will describe simple and relatively robust methods for metabolically radiolabeling proteins in preparation for separation by gel electrophoresis followed by detection by autoradiography.
KeywordsLabel Amino Acid Radiolabeled Amino Acid Glass Fiber Disk Medium Amino Acid Dialyze Fetal Calf Serum
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