Using Shampoo for Blots?
svetlov at oncology.wisc.edu
Mon Nov 30 15:40:07 EST 1998
In article <73rvgo$15m2$1 at news.doit.wisc.edu>,
klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu (Dima Klenchin) wrote:
> Well, sure, I mean, of course, why not, obviously? If you are willing to spend
> a lot of money to get results that are inferior to cheaper freshly prepared
> gels, then I see no reason not to use precast gels. Frees some time for
> Usenet reading too :-))
It seems to me that casting one's own gels does not necessarily preclude
extensive Usenet browsing either <G>.
> The simple fact that other things being equal, fresh gels with real
> stacker _always_ resolve better.
Well, we seem to agree that pre-cast are not entirely unusable now? Good.
X-ray diffraction is a better way to determine the structure of a protein,
but in many cases CD would be enough and in some - like monitoring
un/refolding - actually is much more usable technique. Similarly HPLC or
mass-spec in many instances would have much greater resolution than even
your best PAGEs, and yet I'm not insisting that you abandon your archaic
habit of separating proteins in polyacrylamide. As I said, for most part
precasts work fine in applications we want them to, moreover, the Man (Dick
that is) is a big fan of these things. Meaning that a) we are buying them
for our own work and b) his CSHL protein course is done on Novex gels.
Judging by those big cheeses that take the course soon you would not find a
lab without a good supply of Novex. Moreover, our own subversive activity
resulted in Casper's and Ross's labs getting hooked on Novex... Having
done my fair share of casting all kinds of gels from protein to 1.5 m long
sequencing gels I can appreciate an opportunity not to deal with this stuff
anymore, especially since I have to do all the rest of my stuff from
sequencing and cloning to strain construction alone. And to read Usenet...
Hey, I even switched to automated sequencing now - what about you? This
sequencing is also more expensive than good ole P32 or S35 kits... BTW, the
dude who insisted on making 1.5 m long sequencing gels also had his ideas
about what works better.
> We go through a lot of gels for many
> trivial and pathetic reasons, and couldn't afford precast anyway.
> Myself, I prefer to pour my own also because I like being able to vary
> %, width, number of wells, etc depending on exact application.
Most of these variations are also available in precasts, BTW. The point,
however, is that better ot worse in this as in many other cases is very
much context-dependent. For example, in my own work these PAGEs is a last
step in a work which is most intensive at the stages of cloning, protein
engineering, strain construction and protein-complex purification. PAGE is
essentially reduced to the means of getting a western and if not for the
retrogrades-editors many of the conclusions about assembly of the mutant
subunits could be as easily made based on a dot blot. The rest is fashion
and/or personal taste.
Vladimir Svetlov, PhD
McArdle Lab for Cancer Res.
1400 University Ave.
Madison, WI 53706
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