Message-Id: <9701152356.AA25450@MIT.MIT.EDU>
Date: Wed, 15 Jan 97 18:57:34 EST
From: cook@mit.edu (richard f. cook)
Subject: PROT SEQ: Radiosequencing
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Mark,
When we radiosequence, we use the arylamine kit from Millipore and
follow the instructions in the kit. (Millipore's Sequelon AA membrane,
Aryl-amino derivatized PVDF(Millipore # GEN920033, 20 discs, $200).
We use the standard plumbing configuration on the model 477 and stick
the model 120 injection needle into a fraction collector (Gilson FC80)
which is wired up to the auto zero event lead in the back of the 120 to
make it jump. We extract with neat TFA. Yields are about 30-35% which I
think is what most people get and you can find the rest of the counts on
the filter paper.
Has anyone ever come up with a good explanation as to why so much sample
remains permanently bound to the paper or permanently stuck in the gel
slice?
Dick Cook
MIT
-----------------------
We have been attempting to do some radiosequencing on our ABI 477's.
The proteins of interest are dual labeled: 3H-leucine and
35S-methionine.
I have been using ATZ cycles (after replumbing line #22 from the B block
to
line #11 on the A block), collecting the entire fraction, and counting
it.
I also reset the instrument before starting the sequence, and activated
valve #22 to move the fraction collector to the correct position.
Our results have been dissapointing. The samples are hot enough,
but
the peaks that I am getting are only 10 or 20 dpm's. In the past we
have
successfully gotten good radiosequence results with our Beckman
"spinning
cup" sequencer, but never have with the ABI instruments. I am confident
that the 477 sequencer is performing well with cold sequencing. Can
anyone offer any suggestions? I am not sure if the problem is sample
related, and will be trying to radiosequence a labeled standard.
Thank you in advance for your consideration of my problem.
Mark K. Garfield
Laboratory of Molecular Structure
NIAID/NIH
Rockville, MD
tel: 301-496-3786
email: MG102K@NIH.GOV