From: Michael Morris <michaelm@pharm.usyd.edu.au>
  To  : Joel Mackay <j.mackay@biochem.usyd.edu.au>
  Date: Tue, 10 Oct 2000 13:07:02 +1000

Re: negative XL-A data

Dear Joel,

(i) We have experienced the opposite - increased oxidation of DTT in the
sample side compared to the solvent side resulting in a raised baseline for
the sample. Presumably this is because the presence of protein in the sample
increases the rate of oxidation of DTT. The problem lies with DTT's maximum
wavelength of absorption moving from ~350 nm in its reduced form to ~280 nm
in the oxidised form. We have not, to my knowledge, had increased oxidation
of DTT in the sovent side compared to the sample side resulting in a
negative baseline. Your problem may lie with a mismatch between buffer and
sample (e.g., if you have not dialysed one against the other) or perhaps the
buffer containing DTT was stored for a while with a large headspace of air
above it.

(ii) If you ask the XL-A to do a series of scans at a single wavelength over
several cells or over a period of time you should not experience
difficulties. However, if you are switching between wavelengths then the
problem you have described will occur for the reasons you state.

Yours, Michael



At 09:47 10/10/2000 +1000, you wrote:
>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/
>****************************************************************
>
>
__________________________________________________________________

Michael Morris
Senior Lecturer
Pharmaceutical Chemistry	Tel:	+ 61-2-9351-2359
Faculty of Pharmacy A15	  	Fax:	+ 61-2-9351-4391
University of Sydney 2006	Email:	michaelm@pharm.usyd.edu.au 
Australia			
__________________________________________________________________