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<TITLE>RE: [RASMB] new reductant profiles online</TITLE>
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<P><FONT SIZE=2>Hi all,</FONT>
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<P><FONT SIZE=2>The use of TCEP may be particularly useful when conducting unfolding type experiments (with reductants) in the pseudo-absorbance (or absorbance mode for that matter) mode. Since these SV experiments typically run a bit longer (18 - 24 h), the oxidation of DTT at pH 7, in our experience, can be significant and potentially problematic for analysis at 280 nm.</FONT></P>
<P><FONT SIZE=2>Best,</FONT>
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<P><FONT SIZE=2>Yatin</FONT>
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<BR>
<P><FONT SIZE=2>-----Original Message-----</FONT>
<BR><FONT SIZE=2>From: rasmb-bounces@server1.bbri.org [<A HREF="mailto:rasmb-bounces@server1.bbri.org">mailto:rasmb-bounces@server1.bbri.org</A>] On Behalf Of Leech, AP</FONT>
<BR><FONT SIZE=2>Sent: Friday, September 07, 2007 1:55 AM</FONT>
<BR><FONT SIZE=2>To: RASMB</FONT>
<BR><FONT SIZE=2>Subject: Re: [RASMB] new reductant profiles online</FONT>
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<P><FONT SIZE=2>Hi all,</FONT>
</P>
<P><FONT SIZE=2>I'd like to echo Tom's point that the oxidation changes depend on the</FONT>
<BR><FONT SIZE=2>protein as I have seen this too and it nullifies the idea of having</FONT>
<BR><FONT SIZE=2>a "reference" cell to quantify the effect...</FONT>
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<P><FONT SIZE=2>> Hi-</FONT>
<BR><FONT SIZE=2>> The rate of oxidation depends, too, on the presence of protein and</FONT>
<BR><FONT SIZE=2> > even on the specific protein. I've seen protein samples that have</FONT>
<BR><FONT SIZE=2>> both faster and slower oxidation than solvent alone.</FONT>
<BR><FONT SIZE=2>> Best wishes,</FONT>
<BR><FONT SIZE=2>> Tom </FONT>
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<P><FONT SIZE=2>...and agree with Borries that this causes a particular problem for</FONT>
<BR><FONT SIZE=2>equilibrium runs - especially if the redox changes involve aggregation</FONT>
<BR><FONT SIZE=2>of the protein, so you have curvature changes convolved with baseline</FONT>
<BR><FONT SIZE=2>shifts and everything gets a bit qualitative. Sometimes you just have</FONT>
<BR><FONT SIZE=2>to go back to the user for a "full and frank discussion" about sample</FONT>
<BR><FONT SIZE=2>prep!</FONT>
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<P><FONT SIZE=2>Best regards,</FONT>
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<P><FONT SIZE=2>Andrew</FONT>
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<P><FONT SIZE=2>Borries Demeler wrote:</FONT>
<BR><FONT SIZE=2>> Peter,</FONT>
<BR><FONT SIZE=2>> </FONT>
<BR><FONT SIZE=2>> Thanks for the plot, I will add your data to our website. I agree, the</FONT>
<BR><FONT SIZE=2>> pattern looks *quite* different in TRIS buffer (and less problematic). So</FONT>
<BR><FONT SIZE=2>> your mileage may vary and absorption patterns could differ significantly</FONT>
<BR><FONT SIZE=2>> in different pH, ionic strength, buffer, and protein conditions. Clearly,</FONT>
<BR><FONT SIZE=2>> at the high pH conditions where the reductants were compared, DTT even at</FONT>
<BR><FONT SIZE=2>> low concentration may not be a good choice. I'm also not too sure I would</FONT>
<BR><FONT SIZE=2>> like to have to worry about drifting baselines, especially in equilibrium</FONT>
<BR><FONT SIZE=2>> experiments where you want to maintain as large a dynamical range as</FONT>
<BR><FONT SIZE=2>> possible, and where you have longer run times. I guess the lesson here is</FONT>
<BR><FONT SIZE=2>> to try it out on your particular system if background absorbance matters.</FONT>
<BR><FONT SIZE=2>> </FONT>
<BR><FONT SIZE=2>> -Borries</FONT>
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<P><FONT SIZE=2>-- </FONT>
<BR><FONT SIZE=2>Dr Andrew Leech * Laboratory Manager</FONT>
<BR><FONT SIZE=2>Technology Facility * Molecular Interactions Laboratory</FONT>
<BR><FONT SIZE=2>Department of Biology (Area 15) * Tel : +44 (0)1904 328723</FONT>
<BR><FONT SIZE=2>University of York * Fax : +44 (0)1904 328804</FONT>
<BR><FONT SIZE=2>PO Box 373, York YO10 5YW * Email : apl3@york.ac.uk</FONT>
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