From: LEUNG, IRIS K [PHR/1005] <iris.k.leung@stl.monsanto.com>
  To  : ">"'Joel Mackay'" , rasmb@alpha.bbri.org
  Date: Tue, 10 Oct 2000 10:18:11 -0500

RE: negative XL-A data

Hi Joel,

We have been doing some equilibrium runs with dialyzed samples in the
presence of 1 mM DTT in 25 mM Hepes, 100 mM NaCl,pH 7.5, and observed
an
increase in A280 value in the sample cell over 6 days at 15°C. We
then
repeated the run with 5 mM TCEP (neutral pH, purchased from
Pierce)instead
of DTT in the buffer but also observed a even larger increase in A280
over
the same time period. Prior to the run, we checked that the absorbance
of
buffer alone (containing TCEP)was 0.004 to 0.009 from 280 nm to 260 nm
in a
1 cm path. 

We proceeded to investigate if 5 mM TCEP alone in 25 mM Hepes,pH 7.5,
causes
any change in the absorbance spectrum. After 6 days at room temp., we
observed that TCEP which has been exposed to air (5 mL of buffer stored
in a
capped 15 mL polypropylene tube)gave a peak at 265 nm with an
absorbance of
0.38 (in 1 cm path) compared to 0.05 value observed for the same buffer
stored in a dessicator purged with argon and evacuated several times.
At 2mM
[TCEP], the A265nm value was about 0.2 for the buffer exposed to air.
We
have not tried TCEP with other neutral pH buffers. I would be
interested in
hearing from others who have used TCEP to find out if this phenomenon
is
unique to Hepes. In particular, for those who found TCEP helpful with
their
AUC experiments, would it be possible to expand on the conditions used?

thank you,
Iris
-------------------

Iris Leung,
Discovery Research,
Pharmacia, (formley Searle/Monsanto)
700 Chesterfield Parkway,
Chesterfield,
MO 63198
(636)-737-5846
email: iris.k.leung@monsanto.com



-----Original Message-----
From: Joel Mackay [j.mackay@biochem.usyd.edu.au]">mailto:j.mackay@biochem.usyd.edu.au]
Sent: Monday, October 09, 2000 6:48 PM
To: rasmb@alpha.bbri.org
Subject: negative XL-A data


hi all,
 From time to time, when we run standard equilibrium scans on protein 
samples, we see that the 280 nm scans dip down to negative values at
the 
meniscus end of the cell. We were thinking that this might be due to
having 
DTT in the buffer, and the DTT (for some reason that is not clear to
me) is 
becoming oxidized more in the reference cell than the sample cell. Have

other people seen this sort of effect?
Does this explanation sound plausible?
If not, does anyone have any other ideas?

Actually, while i am 'here', we also sometimes see the scans 'jump
around', 
such that there are two distinct sets of overlapping scans in the one
cell. 
I have explained this effect to myself by supposing that the
monochromator 
is not selecting exactly the same wavelength each time it goes to that 
cell. The same questions as above apply here!

Thanks in advance,
joel

PS My apologies if these are old chestnuts that have been answered many

times in the past - i have only been in this game for a couple of
years...
*****************************************************************
Dr Joel Mackay
ARC Research Fellow
Department of Biochemistry
University of Sydney,
Sydney, NSW 2006
Australia
ph +61-2-9351-3906
fax +61-2-9351-4726
WWW: http://www.biochem.usyd.edu.au/staff/mackay/
Sydney Protein Group Website:
http://www.biochem.usyd.edu.au/spg/
****************************************************************