From: Jim.Bloom.B@bayer.com
  To  : -         (052)rasmb(a)bbri.harvard.edu <rasmb@bbri.harvard.edu>
  Date: Wed, 12 Feb 1997 12:00:21 -0500

Difference sedimentation

I would very much appreciate some advice!!  I have been interested in the
effect of DTT on the structure of my protein and have determined the
sedimentation coefficient of the protein with and without various amounts of
DTT and observed a small decrease in S which makes sense to me.  I did many
repeat runs but the difference in S was so small that the results weren't
terribly convincing so I tried the Schachman difference technique (with the
E-mail help of Tom Laue-thanks Tom!!!) by which I mean protein in one sector
and protein plus DTT in the other sector of the same cell with the same volume
in each sector.  I got very nice results with this technique and am generally
happy except for one thing...I found it very very difficult to get the two
menisci exactly matched!  some times they would be ok and sometimes slightly
off and the data would be no good.  I tried several pipetting techniques and am
convinced that I am within one microliter out of 400, yet eyeballing the
menisci's seemed to show they were not identical yet in the XLA at speed
(60,000) sometimes they would be nearly identical and sometimes I would see a
gaussian right off the bat...so I tried filling exactly and then adjusting by
eyeball and then things were worse.  Maybe all of this is confusing but what's
up?? are the sectors somehow distorted or not exactly the same??  Does one
sector get distorted at speed diferently than the other or am I just all
thumbs??  I really love this technique both in theory and in practice but when
one in three or two in three of your runs aren't any good one gets tired and
gives up!!  Any advice?

Jim Bloom
Bayer Corp.
Berkeley
510-705-5301
Fax:  510-705-4940