Protein Protocols Protein Protocols

Carbohydrate Electrophoresis 855
Place samples in a dessicator flask with a beaker containing a small amount of P2O5. Attach the dessicator directly to the pump without a cold-trap—any water remaining in
the sample will be trapped by the P2O5. 3. Open a fresh ampoule of anhydrous hydrazine O-linked cleavage reagent. Add 50 µL of
hydrazine to the dried sample using a glass transfer pipet, or a positive displacement
capillary pipet (metal or plastic should not be used). Resuspend the dried sample. Overlay
the sample with dry nitrogen and cap tightly. Hydrazine is very hygroscopic. Discard
unused hydrazine according to your hazardous waste regulations. Do not reuse.
4. Incubate samples for 3 h in a sand bath or dry heat block set at 60°C (do not use a water
bath) to release O-linked oligosaccharides (higher temperatures may result in the degradation
of O-linked sugars or in the release of non-O-linked sugar chains, such as N-linked
sugars, from the sample if they are present).
5. Dry samples in vacuum evaporator on low heat setting.
3.4.3. Re-N-Acetylation Procedure
1. Add 30 µL of Re-N-acetylation buffer to the dried pellet from step 5 in Subheading 3.4.2.
2. Resuspend by vortexing. Spin 2 s in a microfuge.
3. Add 2 µL of re-N-acetylation reagent to the solution. Mix well. Spin 2 s in a microfuge.
4. Incubate tubes on ice for 15 min.
5. Following the 15 min incubation, stop the reaction by adding 60 µL of the desalting resin.
Desalting resin is prepared by adding 0.5 g of Dowex AG50X8 to 0.7 mL water.
Invert or vortex the resin immediately prior to removing the 60 µL for each tube.
Incubate the resin with the sample at room temperature for 5 min mixing by placing the
tube on a shaker or by continuously inverting the tube to keep the resin suspended.
6. Briefly centrifuge to pellet the resin, remove the supernatant (save supernatant) and wash
the resin 2× with 120 µL of water for 2 min each (save supernatant).
7. Combine the resin supernatants in a 1.5-mL microcentrifuge tube.
8. Dry the supernatants in a vacuum evaporator on low heat setting.
3.5. Labeling Oligosaccharides
3.5.1. Preparation of Samples and Standards
If quantitation of the oligosaccharide bands in the samples is required, then one must
compare the intensity of an internal standard (e.g., maltotetraose) band with the
intensity of sample bands and it is, therefore, essential that the standard is present on
each gel used (see Note 5 for preparation and use of this material).
3.5.2. ANTS Labeling
1. Prepare the labeling dye as 1 M ANTS in 15% acetic acid (dye solution can be stored in
the dark at –70°C for up to 2 wk).
2. Prepare 1 M solution of NaBH3CN in DMSO and mix well by vortexing until crystals are
completely dissolved (this reducing agent can be stored for 2 wk at –70°C).
3. Add 5 µL of labeling dye to each dried oligosaccharide pellet. Mix well until the oligosaccharide
pellet is dissolved.
4. Add 5 µL of reducing agent. Mix well by vortexing. Centrifuge 5 s in microcentrifuge.
5. Incubate samples at 45°C for 3 h (temperatures higher than 45°C or times longer than
3 h can destroy or modify carbohydrates, e.g., sialic acids). Greater than 90% of the
oligosaccharides are labeled under these conditions. As a convenient alternative samples
can be labeled at 37°C (not 45°C) overnight (or approx 16 h). These latter conditions result
in labeling of >90% of the oligosaccharides (see Note 6).