Unfortunately, PVDF is not a very good membrane to do on-situ digestion.
However, I think there are a couple of possibilities to work on. But to c=
ome
back to your questions:
1) If there are a lot of tryptic sites, and you have run your protein thr=
ough a
denaturation step, I have not seen any protein not being cleaved by tryp=
sin (in
solution, it is even known to chop "insoluble" proteins).
2) so, why don't you see any peptides? Well, the most likely answer is th=
at the
peptides are still bound on the PVDF membrane (that's why you use this ki=
nd of
membrane for N-terminal sequencing, among others), especially if you used=
one of
the high-binding capacity sort. I do not know what kind of digest buffer =
you
used, but in the absence of high concentration of Tween, Triton X-100 or =
some
sort of detergent, you will not extract a meaningful amount of peptides f=
rom a
PVDF membrane. I suppose you have also tested your enzyme on a test prote=
in
(e.g. on the M.W. marker) to make sure that your enzyme was actually acti=
ve ...
In this business, you always want a negative AND a positive control ...
3) Provided you know that the problem lies in the procedure (and not to a=
faulty
enzyme), your peptides then very probably are still there on the membrane=
s. So,
the problem is to retrieve them in a buffer that is HPLC compatible. I s=
uggest
the following action:
a) incubate your membrane (that was chopped in small pieces of 1 mm2) in=
the
minimum amount of a digestion buffer consisting of 15 mM N-ethyl morphol=
ine, 5
mM acetic acid, 1% w/v Zwittergent 3-16, 1-2 =B5g trypsin for a couple of=
hours at
37 C. Spin down for a couple of seconds, collect supernatent. Rinse the m=
embrane
pieces with an equal volume of digest buffer (without trypsin), spin down=
, and
pool both supenatents.
b) At that stage, your peptides should be hopefully in the supernatent. =
Back in
the time I was at the U. of Washington, in Seattle, we tested the procedu=
re
originally devised by Paul Tempst (Sloan Kettering) and obtained very
encouraging results (see Anal Biochem. 1997 247(2): 310-318) on PVDF memb=
ranes.
On HPLC columns, the Zwittergent elutes typically during the wash phase o=
f the
column. CAVEAT: Do NOT use that protocol on a column smaller than a 0.5 m=
m i.d.
especially if you end up with a volume greater than 50 =B5l. The reason:
Zwittergent binds very efficiently on reverse-phase material and will dis=
place
the less-stringent binding peptides. If you feel you have enough material=
, try a
1 mm i.d. column, otherwise a 0.5 mm i.d. column will do (U.V. limit of
detection of peptides in the high femtomole at 214 nm).
Hope that this will help. E-mail me directly if you need more information
Axel
-------------------------------------------------------------------------=
-------
Axel Ducret, Ph.D.
Senior Research Biologist
Merck-Frosst Canada Inc.
Dept. Biochemistry and Molecular Biology
P.O. Box 1005
Pointe-Claire-Dorval PQ H9R 4P8
Canada
tel. + (514) 428-3428
fax + (514) 428-4900