Protein Protocols Protein Protocols

774 Møller and Poulsen
are stained. Nonglycolylated proteins, however, stain weaker, and it is possible to
exclude the staining of nonglycosylated proteins (see Note 1).
A protocol for staining by the cationic dye “Stains-All”, instead of Alcian blue,
combined with silver is described in ref. 13. This method seems to more uniformly
stain both nonglycosylated and negatively charged proteins
The high sensitivity of the methods makes them very suitable for detection of
proteoglycan/glycoprotein in dilute mixed samples, and for characterization of small
amounts of purified materials, for example, by determination of molecular weight
before and after deglycosylation (14,15). In this connection, it is important to note that,
owing to heterogeneity of carbohydrate substitution, highly glycosylated proteins usually
move as diffuse bands or broader smears, so that unambiguous determination of
molecular weight is not always possible.
For more specific purposes, other detection systems are usually employed. Sensitive
detection of glycoproteins can be achieved with lectins or specific antibodies, most
often after blotting onto a membrane (16,17). Proteoglycans can similarly be identified
by antibodies directed toward the core protein or glycosaminoglycan structures (18).
The methods described here are optimized for the small, supported gels used in the
PhastSystem (Pharmacia, Uppsala, Sweden), which can be programmed for automatic
staining resulting in fast and reproducible results. If other systems (larger gels) are
used, generally a longer time is needed in each step (see Note 2).
2. Materials
2.1. Staining of Proteoglycans
All chemicals used should be of analytical grade, and water should be of high
purity (e.g., Maxima from Elga [High Wycombe, UK] or Milli-Q from Millipore
[Bedford, MA]).
1. Washing solution I: 25% ethanol (v/v), 10% acetic acid (v/v) in water.
2. Washing solution II: 10% ethanol (v/v), 5% acetic acid (v/v) in water.
3. Staining solution: 0.125% alcian blue (w/v) (e.g., Bio-Rad [Hercules, CA] no. 161-0401)
in washing solution I. Stir extensively and filter before use.
4. Stopping solution: 10% acetic acid (v/v), 10% glycerol (v/v) in water.
5. Developer stock: 2.5% (w/v) sodium carbonate in water.
6. Sensitizing solution: 5% (v/v) glutardialdehyde in water. (Prepare fresh when required.)
7. Silvering solution: 0.4% (w/v) silver nitrate in water. (Prepare fresh when required.)
8. Developer: Add formaldehyde to the developer stock to a final concentration of 0.013%
(v/v). For example, add 35 µL of a 37% formaldehyde solution to 100 mL of developer
stock, and stir for a few seconds. (Prepare fresh just before use.)
2.2. Staining of Glycoproteins
1. Prepare all solutions described in Subheading 2.1.
2. Fixing solution: 10% (v/v) trichloroacetic acid in water.
3. Washing solution III: 5% (v/v) acetic acid in water.
4. Oxidizing solution: 1% (w/v) periodic acid in water.
5. Reducing solution: 0.5% (w/v) potassium metabisulfite in water.