Protein Protocols Protein Protocols

Lectin Blotting to Detect Glycoproteins 785
Table 2
Glycans O-Glycosidically Conjugated to Proteins
Type of
O-glycosidic linkage Examples of structures
Mucin type NeuAc(α-2,6)GalNAc(α-1,3) Ser (or) Thr
Gal(β-1,3)GalNAc(α-1,3) Ser (or) Thr
The presence of such O-glycosidically linked moieties has been
established for a number of plasma proteins (IgA, IgD,
hemopexin, plasminogen, apoE)
Proteoglycan type [GalNAc(β-1,4)GlcUA(β-1,3)]nGalNAc(β-1,4)GlcUA(β-1,3)
Gal(β-1,3)Gal(β-1,4) Xyl(β-1,3)Ser
They are generally linear polymers made up from repeating
disaccharide units
Collagen type Glc(β-1,2)Gal(β-1,5)OH-Lys
NeuAc, neuraminic acid; GalNAc, N-acetylgalactosamine; Gal, galactose; GlcUA, Glucuronic acid;
Glc, glucose; Xyl, xylose.
Fig. 5. Oligosaccharide inner core common to all N-glycosylproteins.
interest by comparison with a reference 2-D PAGE protein map and to obtain reliable
and reproducible results (30–32).
2. Materials
1. The transfer buffer for protein blotting by the semidry method (see Chapter 40) or by
the tank method (see Chapter 39) is the Towbin buffer diluted 1:2 with distilled water:
12.5 mM Tris, 96 mM glycine, and 10% (v/v) methanol (33).
2. Polyvinylidene difluoride (PVDF) membranes (0.2 mm, 200 × 200 mm) are supplied by
Bio-Rad, the filter papers (chromatography paper grade 3 mm CHr) by Whatman, and the
nitrocellulose membranes (0.45 mm) by Schleicher & Shuell.
3. Blocking solution: 0.5% (w/v) Tween 20 in phosphate-buffered saline (PBS) (34). All
further incubations and washing steps are carried out in the same blocking solution.
4. PBS: 137 mM NaCl, 27 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4. A 10-fold
concentrated PBS solution is prepared, sterilized by autoclaving, and stored at room temperature
for many weeks (10X PBS: 80 g of NaCl, 2 g of KCl, 14.4 g of Na2HPO4, 2.4 g of
KH2PO4. Add H2O to 1 L).
5. Biotinylated lectins are obtained from Boehringer-Mannheim. HRP-labeled Extravidin
(a modified form of affinity-purified avidin) and AP-labeled Extravidin were from Sigma.
6. Two different detection methods are described: the chemiluminescent detection of HRP
activity using the luminol reagent (ECL Kit, Amersham International) and the conventional
colorimetric reaction of AP revealed by nitroblue tetrazolium/bromochloroindolyl
phosphate (NBT/BCIP).
a. Stock solutions of NBT and BCIP (Fluka) are prepared by solubilizing 50 mg of NBT
in 1 mL of 70% (v/v) dimethylformamide and 50 mg of BCIP disodium salt in 1 mL of