From: Borries Demeler <demeler@bioc09.v19.uthscsa.edu>
  To  : Joel Mackay <j.mackay@biochem.usyd.edu.au>
  Date: Mon, 9 Oct 2000 18:50:13 -0500 (CDT)

Re: negative XL-A data

Joel,
I'd be careful with DTT or BME in AUC experiments with absorption
optics (Raleigh IF is OK). Depending on how much you add, there maybe
considerable additional absorbance because of this reductant, which
may shift your total absorbance to a pretty high level. Keep in mind
that the XLA produces the best and most reliable signal between 0.1 and
0.9 OD and adding DTT or other absorbing chemicals may very well shift
you out of that region, and subtracting it out by reference cell isn't
going to help, because you are just subtracting very dark from very,
very dark, ie., not much signal left over.

My suggestion would be to replace reductants like BME and DTT with
TCEP (Tris(2-Carboxyethyl) Phosphine), which is a reductant that 
doesn't absorb, neither in reduced or oxidized forms. This eliminates
a lot of the problems associated with DTT etc., and allows you to
move your absorbance range into a tolerable range.

Hope that helps, -Borries

> 
> 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/
> ****************************************************************
>