From: Boice, Judith <judith_boice@merck.com>
  To  : rasmb@alpha.bbri.org, "'Joel Mackay' <j.mackay@biochem.usyd.edu.au>
  Date: Tue, 10 Oct 2000 09:04:59 -0400

RE: negative XL-A data

Hi Joel,

We have had extensive experience evaluating proteins that are required to
remain under reducing conditions and I can tell you that it is not unusual
for DTT to variably oxidized.  However, in general we have had more problems
with the sample cell values being higher than normal due to protein
catalyzed oxidation rather than reference cells. Either way, DTT can cause
any problems, especially in longer runs.   One reductant that we have used
is Tris Carboxyethyl Phosphine (or TCEP) - we buy it from Pierce.  This
reductant does hlp the absorbance problem although I must admit I have seen
t least one incident where a protein reduced with TCEP appeared to form
dimers compared to DTT.  I don't know if anyone has experienced this as
well.

Regards,


Judith A. Boice, Ph.D.
> h
P.O. Box 2000, RY32-649
Rahway, NJ  07065



> ----------
> From: 	Joel Mackay[SMTP:j.mackay@biochem.usyd.edu.au]
> Sent: 	Monday, October 09, 2000 7:47 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/
> ****************************************************************
>