Biology Reference
In-Depth Information
33. Running conditions may be adjusted to the separation
problem. The run can be further slowed down by prolonging
the 100- and 200 V-steps (e.g., if the sample is known to
contain high amounts of salt). It may also be speeded up by
shortening or omitting these steps, especially in the second
run. The idea of the second run is to refocus the bands below
the sample applications pieces. Alternatively, the 2,000 V-step
of the fi rst run may be prolonged to achieve the recommended
14.9 kV and the second run canceled. Including an additional
low-voltage step at the end of the run may allow a more fl exible
timetable while keeping bands well focused.
34. In case of samples with high salt content or when focusing in
narrow pH gradients it may be advisable to change electrode
strips and/or fi lter paper. This removes components that may
disturb the run, especially when switching to higher voltages.
35. Sometimes IPG strips tend to collect water on their surface
(either upon aging of the strips or due to high protein load).
Partly, this phenomenon can be handled by shortly blotting the
strip surface with fi lter paper (careful—do not contaminate the
strips—the soft gel is quite sticky!) or by turning the strips over-
edge before putting them into the transparent cover for storage.
36. The gel casting cassette has to be fi lled the same way every time
because a more loose or tight packing will infl uence the length
of the separation gel.
37. Pore size of the gels has to be selected depending on the
protein composition of the sample. Besides gradient gels,
homogeneous gels may be used. They can be cast directly in
the casting cassette (closing the bottom outlet and using the
rubber wedge) or singly in the dual casting stand. For these it
is recommended to overlay the gel surface with water (each gel
separately with about 1 mL, carefully overlaying the more
dense gel solution).
38. Meanwhile fi ll the empty gradient maker with water, opening
the connection between the reservoirs. Cover the casting cassette
with Parafi lm to avoid evaporation of the water layer, but not
earlier than 1 h after casting. The gel will have settled then and
begun to polymerize. If you are short of time, leave it uncov-
ered until the next day.
39. Separation gels can be stored in plastic bags in a cold room for
several days. Pour the stacking gel right before use and use the
gel immediately. Otherwise, due to diffusion, the two buffer
systems will mix and affect the quality of your separation.
40. 20-mL glass tubes with screw caps are usually tight enough to
be horizontally shaken on a laboratory shaker. Take care to
insert the strips in a standardized way, e.g., always with the
anodic side fi rst. They can then be easily taken with forceps at
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