[RASMB] new reductant profiles online

[Peter Schuck]

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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