Protein Protocols Protein Protocols

818 Hounsell, Davies, and Smith
3. Method
1. Dry the glycoprotein (100 µg to 1 mg) in a screw-topped vial and resuspend in 100 µL
50 mM NaOH containing 1 M NaBH 4 (see Notes 1 and 2).
2. Incubate at 55°C for 18 h.
3. Quench the reduction by the addition of ice-cold acetic acid until no further effervescence
is seen.
4. Dry the reaction mixture down, and then wash and dry three times with a 1% acetic acid,
99% methanol solution to remove methyl borate.
5. Resuspend the alditols in H 2O, and pass down a 1-mL H + cation exchange resin. The
alditols will not be retained and will elute by washing the column with water.
6. Dry the alditols and resuspend them in 100 µL of 0.2 M NH 4OH.
7. Activate a phenyl boronic acid (PBA) column with 2 × 1 mL MeOH.
8. Equilibrate the PBA column with 2 × 1 mL 0.1 M HCl, 2 × 1 mL H 2O, and 2 × 1 mL
0.2 M NH 4OH.
9. Add the sample in 100 µL 0.2 M NH 4OH and elute with 2 × 100 µL 0.2 M NH 4OH, 2 ×
100 µL H 2O, and 6 × 100 µL 0.1 M Acetic acid. Collect these fractions and test for
monosaccharide (Chapter 108) or combine and analyze according to Chapter 114.
10. Regenerate the PBA column with 0.1 M HCl and 2 × 1 mL H 2O before storing and reactivation
in 2 × 1 mL MeOH.
4. Notes
1. The NaBH 4/NaOH solution is made up