Protein Protocols Protein Protocols

Glycoprotein Detection 761
104
Detection of Glycoproteins in Gels and Blots
Nicolle H. Packer, Malcolm S. Ball, Peter L. Devine, and Wayne F. Patton
1. Introduction
As we become more aware of the significance of posttranslational modifications,
such as glycosylation, in the production of recombinant proteins and in the proteomic
studies of development and disease, techniques for the identification and characterization
of the oligosaccharides attached to proteins need to be established.
After separation of the proteins by either one-diemensional-(1D)- or two-dimensional-(2D)-polyacrylamide
gel electrophoresis (PAGE), the initial step is to identify
which proteins are glycoproteins so that further characterization can proceed. Various
methods have been developed since the early detection of glycoproteins on gels (1) and
blots, with color, fluorescence, and lectin detection now carried out at the analytical
level. The actual level of detection of course depends on the extent of glycosylation of
the protein, as 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 glycoproteins,
both on gels and blots, after separation by electrophoresis. It should be noted
that all the staining procedures currently in use destroy the structure of the carbohydrate
and thus prevent further analysis of the glycan component once the glycoprotein
is visualized.
As an initial characterization step, once the glycoprotein of interest has been located,
a protocol for analyzing the monosaccharide composition on replicate blots of these
proteins is also described.
1. Periodic acid-Schiff staining is a generally useful technique for locating glycoproteins on
gels and nitrocellulose blots, although the sensitivity may not be sufficient for many
applications. Realistically, only 1–10 µg of a highly glycosylated protein can be detected
and the stain is most useful for mucins and proteoglycans. 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.
2. Digoxigenin (DIG)–anti-digoxigenin, alkaline phosphate (AP) labeling is an extension of
the periodic acid–Schiff method above although the sensitivity is much greater (realistically,
depending on the degree of glycosylation, about 0.1 µg of glycoprotein). Glycoproteins
can be detected on dot blots or after Western transfer to membranes such as
nitrocellulose or polyvinylidene difluoride (PVDF). Vicinal (adjacent) hydroxyl groups
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
761