trypsin digestion and structure of protein

[Richard P. Grant]

In article 
<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
> touched.
> 
> 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?

hth,

r

-- 
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 --