Protein Protocols Protein Protocols

844 Wong et al.
3.4. Modification and Activation of NCM
3.4.1. Modification of NCM
1. To modify the NCM (0.2-µm pore size) for adherence of peptides and glycopeptides,
incubate the membrane with 12 divinyl sulfone in dimethylformamide dissolved for 1 h
in 0.5 M NaHCO 3–Na 2CO 3, pH 10.0.
2. Wash the membrane with distilled water and then incubate in 1% aqueous ethylenediamine
for 30 min, followed by a wash with water.
3. Incubate the membrane in 1% glutaraldehyde in 0.5 M NaHCO 3–Na 2CO 3 buffer, pH 10.0,
for 15 min.
4. Wash the membrane with water and air dry. The membrane is ready for use for post-
HPLC tryptic-digest fractions.
3.4.2. Activation of NCM for Glycoprotein Adhesion
1. To hydrophilically activate NCM without modification (0.45-µm pore size), incubate the
membrane in 100% methanol for 5 min.
2. Wash the membrane in distilled water for 1 min and 50 mM Tris–0.15 M NaCl (TBS)
buffer for an additional 1 min and then air dry. The membrane is now ready for loading
intact glycoproteins samples.
3.5. Spotting of Samples on the NCM
1. Spot (1 µL) intact non-protease-treated glycoproteins (total protein concentration ranging
from 2 µg to 10 ng) or HPLC fractions containing peptides and glycopeptides from the
tryptic digests 1 cm apart either onto the activated or modified NCM, respectively (see
Notes 7 and 8).
2. Allow samples to air dry for at least 10 min before identification of glycans (see Note 6).
3.6. Dot-Blot Lectin Overlay Analysis
3.6.1. Digoxigenin-Labeled Lectins
1. Block the surface of the activated membrane by incubating in the blocking solution (supplied
with the kit) for 30 min with gentle shaking (see Notes 9–11).
2. Wash the membrane twice (5 min each) with at least 50 mL of TBS, pH 7.5.
3. Incubate with the respective lectin solutions: Datura stramonium agglutinin (DSA),
Galanthus nivalis agglutinin (GNA), Maackia amurenisis agglutinin (MAA), and Sambucus
nigra agglutinin (SNA) (an aliquot of the digoxigenin-labeled lectin in 50 mM Tris
containing 1 mM MgCl2 and 1 mM CaCl2) for 1 h (see Note 12).
4. After incubation, wash the membrane 3× with TBS (10 min each).
5. Incubate with anti-digoxigenin (polyclonal sheep anti-digoxigenin Fab fragments conjugated
with alkaline phosphatase) (supplied with the kit) in TBS for 1 h.
6. After washing extensively in TBS, develop the membranes using a freshly prepared staining
solution of 4-nitroblue tetrazolium chloride–5-bromo-4-chloro-3-indolyl phosphate
(NBT/X-phosphate) (supplied with the kit) in buffer (0.1 M Tris-HCl, 0.05 M MgCl2, 0.1 M
NaCl, pH 9.5) (see Notes 13 and 14).
7. Stop the reaction by washing the membranes extensively (at least 20 changes) in distilled
water and leave to air dry (see Note 15).
8. Scan the membrane using a Hewlett-Packard flat bed scanner (see Notes 16 and 17).
3.6.2. Biotinylated Lectins
1. Wash the activated or modified membrane twice (5 min each wash), in PBS containing
0.1% Tween 20 (PBS-T) (see Notes 9 and 10).