Protein Protocols Protein Protocols

Sequencing Heparan Sulfate Saccharides 899
16 mL is required. Mix 9.6 mL of T50%–C5% acrylamide stock with 3 mL of 2 M Tris,
pH 8.8, and 3.4 mL of distilled water.
3. Add 10% ammonium persulfate (30 µL) and TEMED (10 µL) to the gel solution, mix
well, and immediately pour into the gel unit.
4. Overlay the unpolymerized gel with resolving gel buffer (375 mM Tris-HCl, pH 8.8,
diluted from the 2 M stock solution) or water-saturated butanol. Polymerization should
occur within approx 30–60 min. The gel can then be used immediately or stored at 4°C
for 1–2 wk.
3.4.2. Electrophoresis
1. Immediately before electrophoresis, rinse the resolving gel surface with stacking gel buffer
(0.125 M Tris-HC1 buffer, pH 6.8, diluted from the 1 M stock solution).
2. Prepare and degas the stacking gel solution (for 5 mL, mix 0.5 mL of T50%–C5%
acrylamide stock with 0.6 mL of 1 M Tris, pH 6.8, and 3.9 mL of distilled water).
3. Add 10% ammonium persulfate (10 µL) and TEMED (5 µL). Immediately pour onto the
top of the resolving gel and insert the well-forming comb.
3. After polymerization (approx 15 min) remove the comb and rinse the wells thoroughly
with electrophoresis buffer.
4. Place the gel unit into the electrophoresis tank and fill the buffer chambers with electrophoresis
buffer.
5. Load the oligosaccharide samples (5–20 µL dependent on well capacity, containing approx
10% [v/v] glycerol or sucrose in 125 mM Tris-HCl, pH 6.8) carefully into the wells with a
microsyringe. Marker samples containing bromophenol blue and phenol red should also
be loaded into separate tracks.
6. Run the samples into the stacking gel at 150–200 V (typically 20–30 mA) for 30–60 min,
followed by electrophoresis at 300–400 V (typically 20–30 mA and decreasing during
run) for approx 5–8 h (for a 16-cm gel). Heat generated during the run should be dissipated
using a heat exchanger with circulating tap water, or by running the gel in a cold
room or in a refrigerator.
7. Electrophoresis should be terminated before the phenol red marker dye is about 5 cm from the
bottom of the gel. (At this point, disaccharides should be 3–4 cm from the bottom of the gel.)
3.5. Gel Imaging
The most effective approach for gel imaging requires a CCD camera that can detect
faint fluorescent banding patterns by capturing multiple frames. Systems commonly
used for detection of ethidium bromide stained DNA can usually be adapted with
appropriate filters as described below (see Note 6).
1. Place a UV filter (UG-1, UG-11, or MUG-2) onto the transilluminator, and fit a 450-nm
blue filter onto the camera lens.
2. Remove the gel carefully from the glass plates after completion of the run and place on the
UV transilluminator surface wetted with electrophoresis buffer. Wet the upper surface of
the gel to reduce gel drying and curling.
3. Switch on the transilluminator and capture the image using the CCD camera. Exposure
times are typically 1–5 s depending on the amount of labeled saccharide (see Note 7).
3.6. Data Interpretation
The sequence of saccharides can be read directly from the banding pattern by interpreting
the band shifts due to removal of specific sulfate or sugar moieties. Figure 1