Message-Id: <9701201600.AA0780@pho903.sbphrd.com>
From: Jacek Mozdzanowski-1
Date: 20 Jan 97 7:57:26 EDT
Subject: proseq
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Larry,
In general there is no difference in sequencer performance for both
tris/glycine and tricine gel systems. The catch is to have efficient
electroblotting. Standard tris-tricine gels contain 0.1% SDS (as well as the
electrophoresis buffer). This _may_ limit protein mobility and decrease the
recovery on PVDF membrane. Specific handling depends on the gel thickness. I
would suggest that you do not presoak the gel in transfer buffer prior to the
transfer if the gel is 1 mm thick (or less). For thicker gels brief rinse may
be helpful. In some cases you will have to prepare transfer buffer without
methanol in order to get satisfactory electroblotting yield. Staining the gel
after transfer helps to determine the best correctional action. All the
precautions valid for tris/glycine system are also valid for tris/tricine
systems. For details refer to "Current protocols in protein science". And
everything works fine as long as the protein is not N-terminally blocked...
But even if it is, you can use protein band for the internal sequencing
(in-situ tryptic digest followed by HPLC peptide map followed by sequencing of
selected peaks). To feel comfortable you want to see about 5 to 10 picomoles
of protein on the PVDF membrane. Amido black is usually recommended to stain
PVDF membranes. The amount loaded to the gel should be larger and depends of
the electroblotting yield. Internal sequencing off PVDF membranes requires
some practice - the lower limit is somewhere between 10 - 20 picomoles.
Working at this level of sensitivity may be - initially - quite demanding.
Jacek Mozdzanowski
Analytical Sciences
SmithKline Beecham