Allozyme electrophoresis & Crustaceans
John H McDonald
mcdonald at strauss.udel.edu
Mon Dec 5 18:28:54 EST 1994
In article <3bvup9$9cj at beta.qmw.ac.uk>, K.A.Foley <bt3027 at qmw.ac.uk> wrote:
>HELP!! I'm doing a population study on a mudflat amphipod ( Corophium
>volutator ), using allozyme electrophoresis . The protocols I'm using
>were developed as a rough guide for insects - there seems to be no
>previous crustacean study of this kind. Can anyone suggest a good
>staining recipe with appropriate running buffer...I'd like to see
>bands instead of smears!
If you're seeing smears instead of nothing, that suggests that your stains
are working, you just need better resolution. The ancient art of allozyme
electrophoresis has always included a good bit of trial and error with the
buffer system. Your favorite enzyme may resolve well in buffer X for some
species, and be a big smear in buffer X from related species. You'll just
have to make up four or five buffers and try all your enzymes on all of
them. Chapter 4 in "Molecular systematics," edited by D.M. Hillis and C.
Moritz, is a good place to look for recipes. I'd recommend their
lithium-borate/tris-citrate, tris-borate EDTA I, tris-citrate II,
tris-citrate/borate, and tris-maleate-EDTA, for starters.
Another cause of smeariness might be digestive enzymes in your
amphipods, which can chew up your enzymes once you grind the bug up. You
should try removing and grinding up some bits without digestive tissue,
such as legs or antennae. However, in my experience with amphipods, this is
only necessary for a couple of enzymes.
John H. McDonald
Department of Biology
University of Delaware
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