Protein Protocols Protein Protocols

Lectin Blotting to Detect Glycoproteins 783
Fig. 4. Glycoprotein blot pattern of 2-D PAGE separation of plasma proteins (120 µg) probed
with SNA (specific for neuraminic acid linked α-2,6 to galactose) and revealed using chemiluminescence
(30 s film exposure). 1, Complement 3 α-chain; 2, transferrin; 3, IgM µ-chain;
4, hemopexin; 5, IgA α-chain; 6, fibrinogen γ-chain; 7, haptoglobin β-chain; 8, fibrinogen
β-chain; 9, IgG γ-chain.
for the monosaccharides or their derivatives. They do not have an absolute specificity
and therefore can bind with different affinities to a number of similar carbohydrate
groups. Because lectin binding can also be affected by structural changes unrelated to
the primary binding site, the results obtained with lectin-based methods must be interpreted
with caution (2).
Despite these limitations, lectin probes do provide some information as to the nature
and composition of oligosaccharide substituents on glycoproteins. Their use together
with the blotting technique provides a convenient method of screening complex protein
samples for abnormalities in the glycosylation of the component proteins. Lectin
blotting requires low amounts of proteins and is easy to perform; it is therefore particularly
indicated for analysis of biological samples.
When the lectin blotting method described in Subheading 3. is combined with the
high resolution and reproducibility of 2-D PAGE and with the sensitivity of
enhanced chemiluminescence, it is possible to identify rapidly the glycoproteins of