From: Chin, Christopher <cchin@utmb.edu>
  To  : 'Joel Mackay ' <j.mackay@biochem.usyd.edu.au>
  Date: Tue, 10 Oct 2000 11:50:54 -0500

RE: negative XL-A data

Joel,

Beside TCEP, BME as suggested by others, I like to give you another potent
reducing agent to add to your arsenal.  TBP (Tri-n-butylphosphine), Aldrich
Chemical, has very little absorption between 230 to 300nm. I have used this
reagent in the study of protein Sulfhydryls and Disulfides (Analytical
Biochemistry 214, 128-134 (1993)).  I have also tried it with AUC and it
works very well. 


-------------------------------------
Christopher Chin
Manager
Sealy Center for Structural Biology
HBC&G, 5136 MRB. rt1055 UTMB
cchin@utmb.edu, 409-772-1693, efax 708-585-1920
-------------------------------------





 


-----Original Message-----
From: Joel Mackay
To: rasmb@alpha.bbri.org
Sent: 10/9/00 6:47 PM
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/
****************************************************************