[RASMB] DTT for equilibrium runs?

[John Correia]

Debby
 
DTT can alter the signal/noise by increasing the absorbance in the
sample buffer, but it can also change over time as the reduced/oxidized
ratio changes thus shifting the baseline.  The reported advantage of
TCEP is it does not absorb at 280 nm - plus is has a pH independent and
stronger reducing power.  In practice both DTT (DTE) and TCEP seem to
have altered absorbance in a protein dependent manner - its as thought
the protein facilitates a reaction.  The major player is O2 and thus
using fresh buffer, degassing and working at lower temperatures for
slower reaction rates help.  Reducing the concentration may be the best
course of action.  Use 0.1 - 0.2 mM instead of 1-2 mM.  The need to use
reducing agent is of course system dependent & worth checking.
 
A search of the RASMB will find these same suggestions from others in
the past.
 
-------------------------------------------------------------------
 Dr. John J. "Jack" Correia
 Department of Biochemistry
 University of Mississippi Medical Center
 2500 North State Street
 Jackson, MS  39216
 (601) 984-1522                                 
 fax (601) 984-1501                             
 email address: jcorreia at biochem.umsmed.edu     
 homepage location: http://biochemistry.umc.edu/correia.html
 dept homepage location:    http://biochemistry.umc.edu/
-------------------------------------------------------------------
 
 


>>> Debby Pheasant <pheasant at MIT.EDU> 06/06/06 12:59 PM >>>

Hi, everyone,

I just heard recently that it's not good to have DTT in your buffer for

doing equilibrium runs.  Can someone explain to me why that would be? 
Or 
share stories of DTT use?

Thanks very much,

Debby

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