Protein Protocols Protein Protocols

196 Ünlü and Minden
in the same groove as the gel, and the current is started promptly, most of the sample will
still go in.
17. For 13 cm long dry strips, 20 kVh total also produces acceptable results in most cases.
18. A small disposable Petri dish makes a great receptacle for equilibration. Wrap the gel
around the inside of the dish, with the gel side pointing in. The dishes may also be marked
to easily keep track of the identities of multiple gels.
19. Before starting to pour the gradient gels, make sure the gradient maker chamber is free of
polyacrylamide pieces. The tube and mixing channel can easily be blocked by small pieces
of gel. Working fast, four gels can be poured one after the other. No washing of the chamber
in between gels is necessary.
20. The 8 h is to allow the polymerization reaction to go to completion. In a crunch, this time
may be shortened to as little as 1 h, but be aware that side chain and N-terminal modification
by acrylamide then becomes more of a problem. Gels may be allowed to polymerize
as long as overnight provided that the butanol layer is increased so that it does not dry out.
We have also stored gels at 4°C for up to 24 h.
21. If the IEF gel has dried, this may cause a problem as the gel has a higher tendency to stick
to the glass and getting torn. Should this start happening, try wetting the IEF strip a little
with equilibration buffer II.
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