[RASMB] varying temperature during an equilibrium run

[Karl Maluf]

Frank, keep in mind that many buffers show a strong temperature dependence
to the pKa.  It is often not possible to maintain identical buffer pH as you
change the temperature, resulting in difficulty with data interpretation;
i.e. is the weaker association observed at higher pH a result of changes in
buffer pH? Karl

 

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From: rasmb-bounces at list.rasmb.org [mailto:rasmb-bounces at list.rasmb.org] On
Behalf Of John Philo
Sent: Friday, October 25, 2013 8:39 AM
To: 'Frank Niesen'; rasmb at rasmb.org
Subject: Re: [RASMB] varying temperature during an equilibrium run

 

Frank, I haven't actually done that type of protocol, but in principle it
should work (assuming the protein is stable enough). My comment though is
that if the association is weaker at higher temperatures you want to do the
higher temperature first and then the lower (for the same reason you always
want to go up in speed for a multi-speed SE sequence). It takes much longer
to reach equilibrium when the change (temperature or speed) will produce a
gradient that is less steep than when the gradient increases.

 

John

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From: rasmb-bounces at list.rasmb.org [mailto:rasmb-bounces at list.rasmb.org] On
Behalf Of Frank Niesen
Sent: Friday, October 25, 2013 7:47 AM
To: rasmb at rasmb.org
Subject: [RASMB] varying temperature during an equilibrium run

Hello, 

 

I was wondering if anybody has experience with changing the temperature in
an equilibrium run at given speed, to investigate temperature-dependent
strength in association? Is it feasible/sane to record data at equilibrium
at, e.g., 20C, and then to change the temperature to, e.g., 40C, and to hope
to get data for equilibrium and record once more?

 

In my case, the association seems to weaken substantially with temperature
and I observe a weaker curvature at the higher temperature. For a third
step, going back to the original temperature, I programmed the run, but made
two mistakes with it - firstly to not allow sufficient time for the
equilibrium and, secondly, to somehow got the "stop AUC after last scan"
checked :o(

As result, I am looking at a scan that doesn't resemble the one at lower
temperature, yet it is similar to scans I saw during the equilibration
between the two temperatures at step 2.

 

Has anybody tried something similar? Am I onto something believable/real, or
shouldn't waste any more precious sample on a proper repeat (i.e. separate
runs/samples for each temperature)?

 

Thanks in advance!

Frank

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