Date: Wed, 15 Jan 1997 14:51:27 -0800 (PST)
From: Ken Walsh <walsh@u.washington.edu>
Subject: Re: Radiosequencing
In-Reply-To: <v03007800af02f9117d38@[128.231.233.117]>
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Mark
Are you spiking your sample with plenty of cold protein so that
non-specific (adsorption on surfaces?) losses do not remove most of the
radioactive PTH aa? We used an excess of antibodies to act as scavengers
in this way. At that time, we also collected our product in fraction
collector tubes pre-spiked with a cold PTH-aa mixture to "protect"any hot
PTHaa during subsequent HPLC.
Ken Walsh
Dept Biochem
Univ Washington
On Wed, 15 Jan 1997, Mark Garfield wrote:
>
> 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
>
>
>