Protein Protocols Protein Protocols

Detection of Glycosylated Proteins on SDS 773
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Staining of Glycoproteins/Proteoglycans in SDS-Gels
Holger J. Møller and Jørgen H. Poulsen
1. Introduction
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a commonly
employed technique for separation of proteins according to size. Among other
applications, it is used for identification and characterization of proteins on the basis of
molecular weight determinations. In this chapter, staining methods are described that
permit detection of highly glycosylated proteins on SDS gels at levels of a few nanograms.
Proteins with limited glycosylation are most often stained with Coomassie brilliant
blue or, if high sensitivity is needed, with a silver stain ([1], and see Chapter 33). These
stains, however, are far less sensitive when used for detection of highly glycosylated
proteoglycans (protein glycosaminoglycans) or glycoproteins (protein oligosaccharides),
leading to weak staining or even failure of detection. This is presumably the
result of steric interference by the carbohydrates with the binding of silver ions.
Proteoglycans are traditionally stained with cationic dyes, such as Alcian blue or Toluidine
blue (2), that bind to the negatively charged glycosaminoglycan side chains,
whereas more neutral glycoproteins can be detected by some variation of the Schiff
base reaction involving initial oxidation of carbohydrates by periodic acid and subsequent
staining with Schiff’s reagent (PAS) (3), alcian blue (4), or a hydrazine derivate
(5). A protocol for PAS staining of small SDS gels is described in ref. 6. However,
these methods, although useful in many instances, are characterized by low sensitivity,
generally requiring microgram amounts of protein for detection. At the same time, they
are carbohydrate specific, which means that nonglycosylated proteins are not stained.
The two methods described here are based on silver enhancement of traditional staining
methods for proteoglycan and glycoprotein (7–12), which result in a twofold
increase in sensitivity as compared with alcian blue or PAS alone. In both methods,
Alcian blue is used as the primary staining agent, binding either directly to the
proteoglycans (Subheading 3.1.) or to oxidized glycoproteins (Subheading 3.2.), subsequently
enhanced by a neutral silver staining protocol.
The methods also stain highly negatively charged phosphoproteins (e.g., osteopontin
and bone sialoprotein, rich in phosphate and acidic amino acids) that stain weakly with
ordinary methods (13). Furthermore, both glycosylated and nonglycosylated proteins
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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