Protein Protocols Protein Protocols

Carbohydrate Electrophoresis 853
2. Gel running buffer: 50 mM Tris-glycine (pH 8.2).
3. O-linked cleavage reagent: anhydrous hydrazine, 1 mL amp. Hydrazine is toxic and
flammable; discard ampoule and residual contents after using once; dispose of safely
according to your institution’s regulations.
4. Re-N-acetylation reagent: acetic anhydride.
5. Re-N-acetylation buffer: 0.2 M ammonium carbonate, pH 9.4.
6. Desalting resin: Dowex AG50X8.
7. Tracking dye: Mixture of Thorin 1, Bromphenol blue, Direct red 75, and Xylene cyanole
in water.
8. Sample loading solution: 25% glycerol with Direct red 75 (store at 4°C).
9. Labeling dye: 1 M 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) in 15% acetic acid
(reagent is stable for 2 wk if stored in the dark at –70°C).
10. Reducing agent: 1 M sodium cyanoborohydride (NaBH 3CN) in DMSO (reagent is stable
for 2 wk at –70°C).
11. Distilled-deionized water.
12. Microcentrifuge tubes (2-mL, glass-lined).
13. Assorted pipeting devices including a 0–100 µL capillary positive displacement pipet.
14. Centrifugal vacuum evaporator.
15. Oven or water bath set at 37°C.
16. Sand filled heat block set at 60°C.
17. Vacuum dessicator.
18. Microfuge.
19. Phosphorus pentoxide (P 2O 5).
20. Bovine submaxillary mucin control (optional).
21. Maltotetraose or partially hydrolyzed starch standard (optional).
3. Methods
3.1. Principle
Using fluorescent PAGE, individual oligosaccharides can be quantified to obtain
molar ratios, to obtain degree of glycosylation, and to detect changes in the extent or
nature of glycosylation. Oligosaccharide profiling involves four steps:
1. Release of the oligosaccharides from the glycoprotein enzymatically or chemically;
2. Labeling of the mixture of released oligosaccharides with a fluorescent tag;
3. Separation of the fluorophore-labeled oligosaccharides by PAGE; and
4. Imaging of the gel either on a UV lightbox to obtain qualitative band information or using
a commercial imaging system to determine the amount of oligosaccharide present in each
band and the relative mobility of the bands.
Once separated on the gel, individual oligosaccharide bands can also be purified for
further study.
3.2. Preparation of Glycoprotein
1. Isolate the glycoprotein according to your usual procedures. The sample should be relatively
salt-free and contain no extraneous carbohydrates (e.g., sephadex-purified material
contains large amounts of glucose) (see Note 1).
2. If the volume of the glycoprotein solution required is >100 µL, dry the glycoprotein in a
1.5-mL microcentrifuge tube. Generally 50–200 µg of glycoprotein is required for