Re: Digest/ProtSeq

Dan Crimmins (crimmins@pathbox.wustl.edu)
Mon, 2 Mar 1998 14:59:39 -0600 (CST)

At 12:16 PM 3/2/98 -0500, Axel Ducret wrote:
>Dear Rebecca,
>
>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 =
come
>back to your questions:
>
>1) If there are a lot of tryptic sites, and you have run your protein th=
rough a
>denaturation step, I have not seen any protein not being cleaved by
trypsin (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 t=
hat the
>peptides are still bound on the PVDF membrane (that's why you use this k=
ind of
>membrane for N-terminal sequencing, among others), especially if you use=
d
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 =
from a
>PVDF membrane. I suppose you have also tested your enzyme on a test prot=
ein
>(e.g. on the M.W. marker) to make sure that your enzyme was actually act=
ive ...
>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 membran=
es. So,
>the problem is to retrieve them in a buffer that is HPLC compatible. I =
suggest
>the following action:
>
> a) incubate your membrane (that was chopped in small pieces of 1 mm2) i=
n the
>minimum amount of a digestion buffer consisting of 15 mM N-ethyl morpho=
line, 5
>mM acetic acid, 1% w/v Zwittergent 3-16, 1-2 =B5g trypsin for a couple o=
f
hours at
>37 C. Spin down for a couple of seconds, collect supernatent. Rinse the
membrane
>pieces with an equal volume of digest buffer (without trypsin), spin dow=
n, 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 proced=
ure
>originally devised by Paul Tempst (Sloan Kettering) and obtained very
>encouraging results (see Anal Biochem. 1997 247(2): 310-318) on PVDF mem=
branes.
>On HPLC columns, the Zwittergent elutes typically during the wash phase =
of the
>column. CAVEAT: Do NOT use that protocol on a column smaller than a 0.5 =
mm 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 di=
splace
>the less-stringent binding peptides. If you feel you have enough materia=
l,
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 informatio=
n
>
>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
>
>
>


Rebecca,
        If you still have the blot that gave NO sequence you might be in
luck.  Contrary to what Axel said that PDVF is a poor choice for in situ
digestion (I'm assuming that to be PROTEOYLIC digestion) we have found th=
at
PVDF is an excellent choice for in situ CHEMICAL cleavage particularly if
your goal is to show that (i) sufficient protein is/was available for
sequencing but presumably blocked and (ii) obtain some sequence for prote=
in
i.d.  The yields are not great BUT what else are you going to do with the
previously "sequenced" sample.  I would recommend CNBr as my first choice.
We have been using this procedure since 1988 and almost ALWAYS generate
interpretable data following in situ CNBr because (fortuitously) there is
generally a primary (main) sequence in addition to several minors.  If yo=
u
already have the sequence then this should not be a problem but you could
use something like SEQSORT (Henzel et al., (1996) Techniques in Protein
Chemistry VII, 341-346) for mixture sequences.


Dan L. Crimmins
Washington University School of Medicine
Dept. Pathology/Division of Laboratory Medicine
660 S. Euclid Ave., Box 8118
St. Louis, MO 63110
Phone:  314-454-8514; Fax:  314-362-1461
e-mail:  crimmins@labmed.wustl.edu