Gylcoprotein Detection 341 38 341 From: Methods in Molecular Biology, Vol. 112: 2-D Proteome Analysis Protocols Edited by: A. J. Link © Humana Press Inc., Totowa, NJ Glycoprotein Detection of 2-D Separated Proteins Nicolle H. Packer, Malcolm S. Ball, and Peter L. Devine 1. Introduction The profile of the two-dimensional (2-D) separation of the protein comple- ment (proteome) of eukaryotic cells and tissues typically contains obvious “trains” of spots that differ in pI and/or apparent molecular mass. These are usually isoforms of the same protein and result from a variety of posttransla- tional modifications. There is growing evidence that alterations to the glycosylation of a protein can be correlated with developmental and pathologi- cal changes; these changes can be visualized on the 2-D separation by alter- ations in the pattern of these glycoprotein isoforms. The initial step, once a 2-D separation has been achieved, is to identify which spots are the glycoproteins of interest, so that further characterization can pro- ceed. Various methods have been developed for the detection of glycoproteins on 2-D gels (1) and blots by color and lectin analysis, and these can be carried out at the analytical level. The actual level of detection of course depends on the extent of glycosylation of the protein, since the reagents react only with the carbohydrate moiety. We have chosen to describe here the stains that we have found to be the most useful for visualizing, both on gels and blots, the glyco- proteins separated by 2-D electrophoresis. A protocol for analyzing the monosaccharide composition of these glycoprotein spots is also described. 1. Periodic acid/Schiff staining is a generally useful technique for locating glyco- proteins on gels and nitrocellulose blots, though the sensitivity may not be suffi- cient for some applications. Realistically, 1–10 μg of a highly glycosylated protein can be detected. Periodic acid oxidizes vicinal diols of glycosyl residues to dialdehydes. The aldehydes are then allowed to react with fuchsin (Schiff’s reagent) to form a Schiff base. Glycoproteins stain pink with fuchsin on a clear background.