> Protein Expression Analysis
Two-dimensional polyacrylamide gel electrophoresis
Proteins are resolved by two consecutive, high-resolution separations. The first is isoelectric focusing, which discriminates on the basis of isoelectric point. The second is SDS polyacrylamide gel electrophoresis, which discriminates on the basis of apparent molecular weight. The technique is able to provide information about the expression levels of up to 1500 protein species representing the most abundant constituents of a mixture. It is most effective for proteins of molecular weight greater than 10,000, because smaller proteins are less sensitively detected by staining, and because they separate less well in the second dimension. 2-D electrophoresis is known to perform less well for membrane proteins than for soluble proteins because of solubility characteristics. The technique has the strong advantage that it provides information about changes in posttranslational modifications that affect isoelectric point or apparent molecular weight. Proteins detected during 2-D electrophoresis are readily identified by mass spectrometry after electrophoresis has been performed. (more details)
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