Biology Reference
In-Depth Information
the gel assembly in a cold room to facilitate disassembling.
In either case, the clamps are then removed, and a long thin
blade is introduced carefully below the supporting sheet along-
side the edges of the rubber frame. If the assembly does not
split immediately, the blade may be turned slightly. The gel on
the supporting fi lm should then separate from the glass plate.
It is put immediately into a large glass tray and washed with
dilute glycerol, under constant and very slow shaking.
5. After 1 h the IPG gel is removed from the washing solution,
put on a dry glass plate (backing downwards), fi xed at the sides
with some bulldog clamps, turned to a vertical position and
dried under a cold stream of air with a ventilator or fan. This
usually takes 1-1.5 h (see Notes 27 and 28).
6. Dried gels are stored in a transparent envelope and a plastic
bag at −20°C (see Note 29).
3.3. First Dimension/
IEF
1. The IPG plate is taken out of the freezer and out of the plastic
bag. At the anodal side, there is a 5-mm margin of plastic
support; on the cathodal side, the margin is wider. Strips are cut
over the complete width of the plastic backing. Use an overhead
marker to mark 5-mm-wide strips on the back of the plastic
cover and then cut along the line, as straight as possible (see
Note 30). Use normal, good scissors and cut the gel when it is
still in the transparent envelope. This will protect the sensitive
gel side from damage and fi ngerprints. Cut as many strips as you
plan to include in your experiment. If this is a new gel, discard
the fi rst strip (the quality is not good enough: Usually, edges are
not nicely polymerized and the iso-pH gradient is not straight).
2. The appropriate volume of re-swelling solution is thawed at
room temperature (315 mL per strip). Put a plastic DryStrip
Aligner on a glass plate of equal size (see Note 31). Distribute
315 mL of re-swelling solution in one lane, making the line of
liquid approximately as long as the IPG strip. Put the strip gel
side down onto the liquid, trying to avoid trapping of air
bubbles. Air bubbles will mean “holes” in the re-swollen strip
and disturb the electric fi eld. Proceed in a similar way with all
your strips, taking care not to spill the solution between the
lanes (you may leave every second lane empty). Having this
completed, cover everything with a glass tray or another
container to prevent excessive drying of the incubating strips.
Do not move the container for the fi rst 15 min (this is the time
needed for the strips to take up most of the liquid). Afterward,
you can shift the whole assembly to a quiet place where gel
strips can swell undisturbed for the next 6-7 h.
3. Preparing the samples: Thaw the labeled samples, spin them
briefl y, and mix the ones you want to run on each gel strip.
If necessary, spin again.
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