[RASMB] new reductant profiles online

[Borries Demeler]

Peter,

Thanks for the plot, I will add your data to our website. I agree, the
pattern looks *quite* different in TRIS buffer (and less problematic). So
your mileage may vary and absorption patterns could differ significantly
in different pH, ionic strength, buffer, and protein conditions. Clearly,
at the high pH conditions where the reductants were compared, DTT even at
low concentration may not be a good choice.  I'm also not too sure I would
like to have to worry about drifting baselines, especially in equilibrium
experiments where you want to maintain as large a dynamical range as
possible, and where you have longer run times. I guess the lesson here is
to try it out on your particular system if background absorbance matters.

-Borries


> 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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