Protein Protocols Protein Protocols

762 Packer et al.
in sugars of glycoconjugates are oxidized to aldehyde groups by mild periodate treatment.
The spacer-linked steroid hapten digoxigenin (DIG) is then covalently attached to these
aldehydes via a hydrazide group. DIG-labeled glycoconjugates are subsequently detected
in an enzyme immunoassay using a DIG-specific antibody conjugated with AP. DIG
glycan detection is known to label almost all known N- and O-linked glycans including
GPI anchors.
3. Lectins are carbohydrate binding proteins which are particularly useful in glycoprotein
and carbohydrate analysis as they can be conjugated to a variety of enzymes or haptens for
use in sensitive detection systems. Their specificity can be used to probe for specific structures
in the glycoconjugates. Lectins are usually classified on the basis of the monosaccharides
with which they interact best, but it is important to note that complex
glycoconjugates are generally found to be much better ligands. In addition, the position of
a particular monosaccharide in a glycan chain (i.e., to what it is attached) will affect lectin
binding, so results obtained in lectin binding studies should be treated with caution. For
example, (a) the wheat germ agglutin (WGA) is inhibited most strongly by dimeric
GlcNAc, but in glycoproteins this lectin also reacts very strongly with sialic acid and
peptide-linked GalNAc; (b) the peanut agglutin binds to Gα1,3GalNAc but does not react
when this structure is sialylated. The labeled lectin–carbohydrate conjugate can then be
visualized by enzyme immunoassay in the same way as in (b).
4. Recently a new kit that uses a fluorescent hydrazide to react with the periodate-oxidized
carbohydrate groups on glycoproteins has been released commercially. The fluorescent
tag (Pro-Q Emerald 300) is excited by ultraviolet light and emits at a visible light (green)
wavelength. The fluorescent signal allows an increased level of detection of the glycoproteins
(down to 1 ng of protein) on both gels and PVDF blots, while allowing the subsequent
visualization of the total proteins with another fluorescent stain emitting at a
different wavelength.
5. The monosaccharide composition of a glycoprotein is a useful start to full characterization.
This is obtained by hydrolysis of the separated glycoprotein spots that have been
electroblotted to PVDF, followed by monosaccharide analysis using high pressure anion
exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (2).
In all methods, it is useful to include a glycosylated protein such as transferrin or
ovalbumin in the marker lane as a control.
2. Materials
2.1. Periodic Acid–Schiff Staining
1. Solution A: 1.0% (v/v) periodic acid in 3% acetic acid. Periodic acid is corrosive and
volatile—handle with caution. Be aware of the concentration of periodic acid in the solution
that is being diluted, as periodic acid is only about 50% out of the reagent bottle.
2. Solution B: 0.1% (w/v) Sodium metabisulfite in 10 mM HCl.
3. Schiff’s reagent: A commercial reagent from Sigma Chemical Co. (St. Louis, MO, USA)
may be used or better staining can often be achieved by making fresh reagent:
a. Dissolve 1 g of basic fuchsin in 200 mL of boiling distilled water, stir for 5 min and
cool to 50°C.
b. Filter and add 20 mL of 1 M HCl to filtrate.
c. Cool to 25°C, add 1 g of potassium metabisulfite, and leave to stand in the dark for 24 h.
d. Add 2 g of activated charcoal, shake for 1 min, and filter. Store at room temperature
in the dark.