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<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'>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<o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=navy face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:navy'><o:p> </o:p></span></font></p>
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<p class=MsoNormal><b><font size=2 face=Tahoma><span style='font-size:10.0pt;
font-family:Tahoma;font-weight:bold'>From:</span></font></b><font size=2
face=Tahoma><span style='font-size:10.0pt;font-family:Tahoma'>
rasmb-bounces@list.rasmb.org [mailto:rasmb-bounces@list.rasmb.org] <b><span
style='font-weight:bold'>On Behalf Of </span></b>John Philo<br>
<b><span style='font-weight:bold'>Sent:</span></b> Friday, October 25, 2013
8:39 AM<br>
<b><span style='font-weight:bold'>To:</span></b> 'Frank Niesen';
rasmb@rasmb.org<br>
<b><span style='font-weight:bold'>Subject:</span></b> Re: [RASMB] varying
temperature during an equilibrium run</span></font><o:p></o:p></p>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'><o:p> </o:p></span></font></p>
<p class=MsoNormal><font size=2 color=blue face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:blue'>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 <u>first</u> 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.</span></font><o:p></o:p></p>
<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'> <o:p></o:p></span></font></p>
<p class=MsoNormal><font size=2 color=blue face=Arial><span style='font-size:
10.0pt;font-family:Arial;color:blue'>John</span></font><o:p></o:p></p>
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<p class=MsoNormal style='margin-bottom:12.0pt'><b><font size=2 face=Tahoma><span
style='font-size:10.0pt;font-family:Tahoma;font-weight:bold'>From:</span></font></b><font
size=2 face=Tahoma><span style='font-size:10.0pt;font-family:Tahoma'>
rasmb-bounces@list.rasmb.org [mailto:rasmb-bounces@list.rasmb.org] <b><span
style='font-weight:bold'>On Behalf Of </span></b>Frank Niesen<br>
<b><span style='font-weight:bold'>Sent:</span></b> Friday, October 25, 2013
7:47 AM<br>
<b><span style='font-weight:bold'>To:</span></b> rasmb@rasmb.org<br>
<b><span style='font-weight:bold'>Subject:</span></b> [RASMB] varying
temperature during an equilibrium run</span></font><o:p></o:p></p>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'>Hello, <o:p></o:p></span></font></p>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'><o:p> </o:p></span></font></p>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'>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?<o:p></o:p></span></font></p>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'><o:p> </o:p></span></font></p>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'>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(<o:p></o:p></span></font></p>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'>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.<o:p></o:p></span></font></p>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'><o:p> </o:p></span></font></p>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'>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)?<o:p></o:p></span></font></p>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'><o:p> </o:p></span></font></p>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'>Thanks in advance!<o:p></o:p></span></font></p>
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<p class=MsoNormal><font size=3 face="Times New Roman"><span style='font-size:
12.0pt'>Frank<o:p></o:p></span></font></p>
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