Electrophoretic Analysis of Yeast Proteins
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Polyacrylamide gel electrophoresis (PAGE) of proteins provides a powerful qualitative and quantitative tool for studying yeast protein synthesis. Complex mixtures of proteins can be separated purely on the basis of molecular weight by means of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ( 1 ), or alternatively on the basis of a combination of both molecular weight and electric charge using two-dimensional electrophoresis (2D SDS-PAGE). Two methods of 2D SDS-PAGE have been described, namely isoelectric focusing (IEF) gels ( 2 ) and nonequilibrium pH gradient (NEPHGE) gels ( 3 ). NEPHGE gels give better resolution of basic proteins compared to IEF gels (Fig. 1 ), and this is particularly useful for analyzing yeast proteins that contain many basic amino acids, e.g., ribosomal proteins and histones.
Fig. 1. Two-dimensional SDS-PAGE analysis of proteins synthesized in vivo in the yeast Saccharomyces cerevisiae . (A) Exponentially growing cells pulse-labeled for 10 min with [ 35 S]-methionine, then separated in the first dimension by a nonequilibrium pH gradient gel (NEPHGE), and in the second dimension by 15% SDS-PAGE (SDS). (B) As for (A), except labeled with a [ 14 C]-amino acid mixture. Open arrow heads indicate proteins not labeled with [ 35 S]-methionine. (C) As for (A), except protein samples were run in a first dimension isoelectric focusing gel (IEF). The proteins indicated are: 1, elongation factor EF-1α; 2, the heat shock protein Hsp70 family of proteins; 3, glyceralde-hyde-3-phosphate dehydrogenase.
