[RASMB] new reductant profiles online

[Gokarn, Yatin]

Hi all,

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.

Best,

Yatin


-----Original Message-----
From: rasmb-bounces at server1.bbri.org [mailto:rasmb-bounces at server1.bbri.org]
On Behalf Of Leech, AP
Sent: Friday, September 07, 2007 1:55 AM
To: RASMB
Subject: Re: [RASMB] new reductant profiles online

Hi all,

I'd like to echo Tom's point that the oxidation changes depend on the
protein as I have seen this too and it nullifies the idea of having
a "reference" cell to quantify the effect...

> 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 

...and agree with Borries that this causes a particular problem for
equilibrium runs - especially if the redox changes involve aggregation
of the protein, so you have curvature changes convolved with baseline
shifts and everything gets a bit qualitative. Sometimes you just have
to go back to the user for a "full and frank discussion" about sample
prep!

Best regards,

Andrew


Borries Demeler wrote:
> 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


-- 
Dr Andrew Leech                   *  Laboratory Manager
Technology Facility               *  Molecular Interactions Laboratory
Department of Biology (Area 15)   *  Tel   : +44 (0)1904 328723
University of York                *  Fax   : +44 (0)1904 328804
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