[RASMB] new reductant profiles online

[Tom Laue]

Hi-
The rate of oxidation depends, too, on the presence of protein and even 
on the specific protein. I've seen protein samples that have both faster 
and slower oxidation than solvent alone.
Best wishes,
Tom

Peter Schuck wrote:
> Borries,
>
> commenting on your warning against the use of DTT, it seems the data 
> you show were collected at a pH of 9.4 (!), which is in a pH range 
> where the reaction is considerably faster and maybe even taking a 
> different path than at neutral pH.  In contrast, the time-course of 
> DTT spectral change in Tris pH 7.5 is much slower, and under these 
> more moderate (and more normal for us) conditions even at 5 mM DTT 
> didn't give more than 0.15 OD at 280 nm:
>
> http://analyticalultracentrifugation.com/images/5mMDTT_Tris_pH7p5.pdf
> (data part of the experimental module of our AUC workshop at NIH.)
>
> To see whether DTT or TCEP is the better choice or not, it seems to me 
> that one may have to consider the entire buffer parameters, and it may 
> depend also on the particular protein studied.  In any case, in the 
> past we've done experiments with 2 - 5 mM DTT and it worked just fine 
> for us (of course taking the slightly increasing baseline into account).
>
> Peter
>
>
>
>
> At 04:28 PM 9/1/2007, you wrote:
>> Hi,
>>
>> I added some absorbance profiles for oxidation time courses
>> for 2mM TCEP and DTT to our reductant information page:
>>
>> http://www.cauma.uthscsa.edu/reductants/index.html
>>
>> (Data courtesy of Marek Luczkowski, University of Michigan)
>>
>> The DTT profile shows some clear evidence why DTT should *not* be used
>> for absorbance experiments, even in small concentration. I am not sure
>> if the refractive index changes with oxidation, but if someone has some
>> data on this please send it to me for inclusion in this website. TCEP is
>> much more stable with respect to absorbance profiles during oxidation
>> and preferred for experiments > 240 nm.
>>
>> Regards, -Borries
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-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
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E-mail: Tom.Laue at unh.edu
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