Application of 2D DIGE in Animal Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 392

26. Always use freshly prepared ammonium persulfate solution to
ensure good and reproducible polymerization. Depending on
the brand and the age of your solid chemical you might need
to adjust the amount to achieve good polymerization in appro-
priate time.
27. Do not prolong the washing time of the IPG gel. It tends to
collect even more water then and the gel layer may peel off the
support. When the gel is not well polymerized, it will also tend
to detach from the fi lm, even during a 1 h-wash. Those gels are
hardly ever to be rescued (except if only the very edge is
affected); then it is better to cast a new gel. Also avoid any
mechanical damage of the gel and take care that gel drying
takes place in a dust-free location.
28. Drying the gels should quickly take place in a stream of cold
air. Drying at room temperature without the help of an air
stream takes too long and affects the performance of the gel.
29. Shelf-life for homemade IPG plates is several months. Upon
aging water exudation (“sweating”) of the strips may start,
getting worse with prolonged storage. Although some water
collecting at the surface of IPG strips is not uncommon in
highly loaded gels, larger amounts cannot be tolerated as pro-
teins collect there, too, and are lost for the analysis. In addition,
if the water is not removed, it tends to blur the focused zones
of the strip.
30. Do not worry if the hand-cut strips are not completely straight
or slightly “crooked”. The important thing is that the gradient
in the gels is well-cast. However, not so nicely cut strips have
to be embedded in agarose with some more care, especially
avoiding air bubbles (see Subheading 3.6 ).
31. Alternatively, strips may be swollen in an upright standing re-
swelling cassette in an excess of re-swelling solution. The
assembly is similar to the described gel casting in Subhea-
ding 3.2 , step 1 (see also Note 21). In addition to the rubber
U-frame attached to the glass plate, a U-frame of the same
dimensions is cut from a double-layer of Parafi lm. This compen-
sates for the thickness of the gel support fi lm and allows swelling
of the strips to original thickness. Rehydration in the cassette
needs much more re-swelling solution, but is recommended
when strips are rehydrated overnight, as there is less risk of
drying out and formation of urea crystals.
32. Depending on the geometry of the electrophoresis chamber a
drying-out phenomenon may occur, even urea crystals may
form on the edges or the surface of the strips. This can partly
be overcome by placing a wet piece of fi lter paper (size approx-
imately 3-4×2 cm) on either side of the strips and/or by
including “blank” strips left and right to the sample-loaded
strips (Fig. 2b ).