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<DIV>Debby</DIV>
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<DIV>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.</DIV>
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<DIV>A search of the RASMB will find these same suggestions from others in the past.</DIV>
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<DIV>-------------------------------------------------------------------<BR> Dr. John J. "Jack" Correia<BR> Department of Biochemistry<BR> University of Mississippi Medical Center<BR> 2500 North State Street<BR> Jackson, MS 39216<BR> (601) 984-1522 <BR> fax (601) 984-1501 <BR> email address: <A href="mailto:jcorreia@biochem.umsmed.edu">jcorreia@biochem.umsmed.edu</A> <BR> homepage location: <A href="http://biochemistry.umc.edu/correia.html">http://biochemistry.umc.edu/correia.html</A><BR> dept homepage location: <A href="http://biochemistry.umc.edu/">http://biochemistry.umc.edu/</A><BR>-------------------------------------------------------------------<BR> <BR> <BR><BR><BR>>>> Debby Pheasant <pheasant@MIT.EDU> 06/06/06 12:59 PM >>><BR></DIV>
<DIV style="COLOR: #000000">Hi, everyone,<BR><BR>I just heard recently that it's not good to have DTT in your buffer for <BR>doing equilibrium runs. Can someone explain to me why that would be? Or <BR>share stories of DTT use?<BR><BR>Thanks very much,<BR><BR>Debby<BR><BR>_______________________________________________<BR>RASMB mailing list<BR>RASMB@rasmb.bbri.org<BR><A href="http://rasmb.bbri.org/mailman/listinfo/rasmb">http://rasmb.bbri.org/mailman/listinfo/rasmb</A><BR></DIV></BODY></HTML>