Protein Protocols Protein Protocols

Microassay of Protein Glycosylation 847
Table 2
The Sensitivity Levels of Commercial Lectins on Glycoproteins
by Lectin Overlay Analysis
Asialylated
Bovine
serum Chicken
Lectin Fetuin fetuin albumin ovalbumin RNase B
MAA .10 ng 0.1 µg NB NB NB
SNA 0.1 µg NB NB NB NB
DSA 0.1 µg 0.1 µg NB NB NB
GNA 0.1 µg .01 µg 2 µg 1 µg NB
NB, No binding of lectin detected to sample.
required for lectin overlay. This is a convenient “one-pot” reaction before purification and
detailed quantitative studies of active oligosaccharides can be done.
6. Take care to allow the samples to air dry completely before the second application
on the NCM.
7. To avoid unnecessary cross-contamination between samples, a 96-well dot-blotter (Bio-
Rad) was used instead of spotting the samples directly onto the membrane. Prior to activation
or modification of the NCM, cut the membrane to size for placing onto the dot blotter
cassette. Once the membrane is in a fixed position, apply respective samples as indicated
in Subheading 3.5. After loading samples remove membrane from dot blotter and leave
to air dry. Once dried, mark the membrane with a pencil to indicate the order of the
samples. Also, draw a grid on paper to indicate the location of samples.
8. When applying respective samples onto the membrane, always use fresh Gilson tips for
each sample to avoid contamination.
9. All incubations in the lectin overlay analysis are carried out on a rocking table at room
temperature.
10. During the lectin overlay analysis, use gloves and avoid direct contact of the membrane
with skin. Handle the membrane carefully with a flat-ended twiser to avoid damage.
11. For a clearer background, the blocking solution can be incubated with the membrane for
overnight at 4°C. After, incubate the membrane with the blocking solution for a further
30 min at room temperature prior to washing.
12. For convenience, use large Petri dishes to incubate each membrane with respective lectin
solutions. Make sure that the lectin solution always covers the membrane surface during
incubation.
13. In contrast to the manufacturer’s instructions the staining reaction can be extended until
significant staining could be observed (1–18 h).
14. Standard glycopeptides should also spotted onto the membrane to monitor the progress of
staining.
15. To protect the membrane from dust and damage, place a clear plastic film over the membrane
and store in the dark.
16. Table 2 presents a summary of results of a series of whole non-protease-treated glycoproteins
(at various concentrations) analyzed for lectin specificity and sensitivity by dot-blot
lectin overlay. The results from this assay clearly show that three lectins (MAA, SNA, and
DSA) recognize Bovine fetuin (BF) at high sensitivity protein levels ranging from 10 ng
to 0.1 µg. Asialylated BF (ABF) were detected by lectins MAA and DSA but not SNA. As
expected, all three lectins showed no binding to Bovine serum albumin (BSA), which is
not glycosylated. According to the lectin specificity as published by the manufacturer,