Protein Protocols Protein Protocols

178 Gianazza
the gel. Depending on the temperature and relative humidity, the drying step may take
between 1 and 2 h, and should not be unnecessarily prolonged. It is acceptable, but not
recommended to let the gels dry unattended, e.g., overnight, at room temperature.
9. For barely damp gels, the very last phase of drying may occur in the refrigerator, with the
gel in an open box. If needed, the polymerized gels may be stored overnight in their cassettes
at 4°C before washing.
10. The dry gels may be further processed right away or stored for later use. Dry IPGs may be
kept in boxes at 4°C for several days. For longer periods of time (22), they are sealed in
plastic bags at –20°C.
4. Notes
1. All required stock solutions are orderly aligned on the bench. It is most helpful if their
sequence corresponds to the order in which they are called for by the recipe. This is especially
important for the acrylamido buffers, whose bottles look necessarily alike, whereas
the pK label is in a relatively small print. It is strongly suggested that the recipe be marked
with a ruler and that each chemical be put back in a box after its use. When a bottle is
open, hold its cap upside down on a paper tissue. Make sure you are storing the open
bottles on a flat, stable surface (like a tray): expensive Immobiline may be easily if inadvertently
spilled.
2. Positive displacement measuring devices might be more accurate than air-displacement pipets.
No liquid droplets must adhere to the outside of the pipet tips; for this purpose, it is usually
sufficient to touch the neck of the bottle. For a quantitative transfer of the Immobiline chemicals,
and especially of the basic monomers dissolved in an alcoholic solution, two precautions
should be taken; namely, slow pipeting and rinsing the pipet tips twice with distilled water
after each measurement.
3. It is not customary to prepare large batches of the limiting solutions—the storage conditions
in the mixture are appropriate for none of the chemicals. However, leftover solutions
might be used within a couple of days.
4. In the case where the commercial products were not available, an adequate substitution
for the gel mold may be a plate of plain glass (polished and saturated with silane) together
with a rubber gasket of approx 0.5-mm thickness. The latter may be cut from foils of para-,
silicone, or nitrile rubber, should have a U-shape, and should be ~5 mm wide. Thick
clamps from stationery stores may then be used, but care should be taken to apply the
pressure on the gasket, not inside it.
5. A note of caution for 1-D experiments with radioactive samples. In some cases, the ink gives
a strong positive signal; more commonly, the writing negatively interferes with film exposure.
In these experiments, all marks should then be outside the area of sample application
(where their disturbing appearance may turn into the definition of useful reference points).
6. The major points of caution will be stressed once more below. The flow between the different
compartments must be unhindered by air bubbles between the two vessels of the mixer or by
water droplets in the outlet. No backflow should occur. The pouring should not be too fast to
allow proper mixing in the chamber and to avoid turbulence in the cassette (with a proper
selection of the pressure drop). The solution should evenly flow along the hydrophilic wall of
the cassette (lined with GelBond) and not fall dropwise along the hydrophobic one.
7. A forced ventilation is more appropriate to this purpose than a convection oven in order to
provide even and controlled heating. It should be recalled that the selected temperature is
the one allowing identical incorporation efficiency for all acrylamido buffers, which grafts
into the gel matrix a pH gradient exactly matching the computed course (23).
8. It is sensible to set aside some containers only for this purpose, i.e., to avoid recycling
between gel washing, protein staining, and blot immunodetection. Owing to its high viscosity,
100% glycerol should be avoided as stock reagent in favor of the 87% preparation; ~12