Nonreducing 2-D Polyacrylamide Gel Electrophoresis

Several approaches can be used for identifying 2-DE resolved proteins, including microsequencing, immunoblotting with highly specific antibodies, and mass spectrometry-based technologies (for a review of the latter approaches, see ref. 1 ). In the case of immunoblotting, although this technique has the advantage of being both fast and specific, it is not always straightforward, since many antibodies fail to recognize proteins following acrylamide gel electrophoresis. This is owing to the fact that some monoclonal antibodies (MAbs) recognize only conformational epitopes, which are dependent on the native spatial conformation of the protein, as distinct from sequential determinants, which are dependent on the linear amino acid sequence of a peptide (for a review, see ref. 2 ). In contrast to sequential determinants, conformational determinants are frequently destroyed by reagents employed in electrophoresis (e.g., reducing reagents, SDS, and/or urea) (3 ,4 ). Although electrophoretic methods used for resolving native proteins have been described (5 ), these techniques are not widely used because of technical problems, such as protein aggregation and the wide variance in the effective electrophoretic mobility of proteins in different buffer systems. Moreover, native 2-DE in our hands has proven to be time-consuming compared to conventional reducing 2-DE (4 ).