Protein Protocols Protein Protocols

856 Brandley, Klock, and Starr
6. After labeling, dry the samples in centrifugal vacuum evaporator for approx 15 min or
until the sample reaches a viscous gel stage.
3.6. Electrophoresis
3.6.1. Preparation of a Sample for Electrophoresis
1. Resuspend the dried fluorophore “labeled” oligosaccharide in 5–20 µL H2O. The actual
volume of H2O used to resuspend the sample will depend on the amount of oligosaccharide
present in the sample (start with 10 µL, this will enable the sample to be diluted
further if necessary).
2. Remove an aliquot of the sample (generally 1–2 µL) and dilute it with an equal volume
of sample loading solution. Load the entire aliquot into one lane of a gel. Best results
are obtained by loading 4 µL/lane on a 10 × 10 cm gel with eight lanes.
3.6.2. Electrophoresis
1. For N-linked analysis chill the running buffer to 4–6°C prior to use. For N-linked
analysis perform electrophoresis at a buffer temperature of 5–8°C. All O-linked gels are
run at 15–20°C.
2. For N-linked analysis set up electrophoresis with a recirculating chiller and place the electrophoresis
tank containing a stir bar on a mechanical stirrer. Connect the gel box cooling
chamber to a refrigerating circulator. Turn on the circulator and stirrer and set the coolant
temperature to 5°C.
3. For N-linked analysis pour the precooled running buffer into the electrophoresis tank up
to the appropriate level. The temperature of the buffer should be monitored during the run
using a thermometer inserted through the hole in the lid or other method. The temperature
will probably increase a few degrees during electrophoresis, but should not exceed 10°C.
For O-linked gels the temperature should not exceed 23°C.
4. Determine the number of gels required for the samples prepared. Each gel should contain
eight lanes. The outside lanes should be used for the tracking dye and glucose polymer
standard leaving the six inner lanes for samples and quantitation standard (maltotetraose).
5. Gently remove the comb(s) from the gel(s). To avoid distorting the wells, gently wiggle
each comb to free the teeth from the gel, then lift up slowly until the comb is released.
6. Place the gel cassette(s), one on each side of the center core unit of the gel apparatus with
the short glass plate against the gasket. Be sure the cassette is centered and that the
cassette is resting on the “feet” at the bottom of the apparatus. If only one gel is being run
place the buffer dam on the other side.
7. It is essential that the wells of the gel are thoroughly rinsed out with the running buffer
from the upper buffer reservoir prior to sample loading. This is best accomplished by
using a syringe with a blunt needle (a Pasteur pipet is not recommended because of the
possibility of breakage into the wells).
8. With the core unit containing the gels placed securely on the bench, load samples into the
wells by underlaying the upper buffer. Use flat sequencing pipet tips to load by delivering
the sample to the bottom of each well. Optimal resolution will be achieved by using 4 µL
of sample per lane.
Note: For the most reliable quantitation of oligosaccharide bands the use of a positive
displacement pipet (e.g., Hamilton syringe) is recommended.
9. Load 4 µL of the standard in a lane when prepared as described in Note 5.
10. Load 2 µL of tracking dye in a lane directly from the vial.
11. Load 4 µL of each labeled oligosaccharide sample in a lane. Samples should be diluted 1:1
in the sample loading solution (see Note 7).