trypsin digestion and structure of protein
Richard P. Grant
rpg14 at yahoo.co.uk.invalid
Wed Feb 7 03:43:29 EST 2001
<Pine.SGI.3.96.1010205101602.18097059B-100000 at mole.bio.cam.ac.uk>,
Michael Witty <mw132 at mole.bio.cam.ac.uk> wrote:
> My boss says that if I can digest my protein with trypsin and discrete
> bands appear, rather than total digestion, I can conclude it has some
> structure other than random coil.
Do your boss and my boss know each other? :-)
> Well, my protein is digested to nothing, compared to BSA, which is hardly
> Do we all think that my protein has no ordered structure in solution, and
> is therefore not worth crystallizing? Mike.
Not necessarily. You should do a titration of trypsin conc, say from
0.5 mg/ml down in 4 or 5 fold dilutions to the ng/ml range. You could
also use chymotrypsin, V8, pepsin, thrombin. RT at 20 minutes should do
it, then quench the proteolysis and boil in Laemmli buffer immediately.
It's worth setting up a Hampton screen anyway, at highish (10 - 20 mg/ml
) [protein] - if you get lots of oils then it's likely to have mobile
portions and be difficult to crystallize.
how big is the sucker?
Richard P. Grant MRC Lab of Mol Biol
rpg 'at' mrc-lmb.cam.ac.uk http://www2.mrc-lmb.cam.ac.uk/personal/rpg/
-- I *love* a target-rich environment --
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