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<DIV dir=ltr align=left><SPAN class=254120023-09122010><FONT color=#0000ff
size=2 face=Arial>Lake, regarding the UV damage, remember that pathway is
independent of whether your protein is stable enough in solution at RT to do
equilibrium. Every biopharmaceutical has to be tested for light stability,
so we biotech types tend to be more aware of these issues than academics. A
very common result of UV exposure is irreversible aggregation. Many SE protocols
involve quite a few scans, for example to try to decide whether
equilibrium has been reached, and often those scans have rather high repetitions
at each radial position. If you do have UV damage during an SE experiment,
you are just going to see that the profile keeps changing as you do more scans
(you don't really reach equilibrium). You could easily mistake this as lack of
physical stability, and may not realize it is the absorbance scans themselves
that are causing the problem. I hear that photodamage can vary a lot from one
protein to another, and I'm not saying that in practice this is a common problem
for SE or SV, but it may not be at all obvious if and when it <U>is</U> actually
a problem.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=254120023-09122010><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=254120023-09122010><FONT color=#0000ff
size=2 face=Arial>Here again is an issue where Beckman could easily help us by
finally providing software that scans the 6-channel cells properly and doesn't
needlessly expose all the samples to UV while it is fruitlessly scanning through
regions of charcoal-filled epon.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=254120023-09122010><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=254120023-09122010><FONT color=#0000ff
size=2 face=Arial>John</FONT></SPAN></DIV><BR>
<DIV dir=ltr lang=en-us class=OutlookMessageHeader align=left>
<HR tabIndex=-1>
<FONT size=2 face=Tahoma><B>From:</B> Dr. Lake Paul [mailto:lpaul@purdue.edu]
<BR><B>Sent:</B> Thursday, December 09, 2010 2:47 PM<BR><B>To:</B> John Philo;
'Roy Hantgan'; rasmb@rasmb.bbri.org<BR><B>Subject:</B> Re: [RASMB] radial scans
6 sector cells<BR></FONT><BR></DIV>
<DIV></DIV>John,<BR>I agree to a point. I usually like to see<BR>my menicus
region just in case I sprung a leak. It is always easier to subtract data than
to add it. As for UV damage, I think if you are going to be doing a SE
experiment your protein has to be stable as a rock for long periods of time near
room temperature, so a little UV damage is not going to be fatal to the
experiment. If UV does damage the protein then I would use interference instead.
<BR>Lake<BR><BR>
<DIV id=htc_header>----- Reply message -----<BR>From: "John Philo"
<jphilo@mailway.com><BR>Date: Thu, Dec 9, 2010 5:00 pm<BR>Subject: [RASMB]
radial scans 6 sector cells<BR>To: "'Roy Hantgan'"
<rhantgan@wfubmc.edu>, <rasmb@rasmb.bbri.org><BR><BR></DIV>
<DIV dir=ltr align=left><FONT color=#0000ff size=2 face=Arial><SPAN
class=344114621-09122010>Lake, it seems to me for equilibrium there is no need
to capture any data to the left of the meniscus, or even the meniscus itself.
You'll just have to trim all that region away before you analyze anyway. So the
default start at 5.8 cm should capture everything you will need. On the other
end I find there is no useful (fittable) data beyond ~7.12 cm. Also remember
that UV does damage proteins, and that all three samples (3 channels) get
exposed with each flash, so at some point extra flashes to collect data you
aren't going to use does become harmful.</SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT color=#0000ff size=2 face=Arial><SPAN
class=344114621-09122010></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT color=#0000ff size=2 face=Arial><SPAN
class=344114621-09122010>Roy, you may find it useful to have both a velocity
method and an equilibrium one open. I usually do a quick velocity-mode scan to
be sure everything is loaded okay and to figure out the radial range I want, and
then set up the equilibrium method window based on the quick velocity-mode scan
results.</SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT color=#0000ff size=2 face=Arial><SPAN
class=344114621-09122010></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT color=#0000ff size=2 face=Arial><SPAN
class=344114621-09122010>John</SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT color=#0000ff size=2 face=Arial><SPAN
class=344114621-09122010></SPAN></FONT> </DIV><BR>
<DIV dir=ltr lang=en-us class=OutlookMessageHeader align=left>
<HR tabIndex=-1>
<FONT size=2 face=Tahoma><B>From:</B> rasmb-bounces@rasmb.bbri.org
[mailto:rasmb-bounces@rasmb.bbri.org] <B>On Behalf Of </B>Lake
Paul<BR><B>Sent:</B> Thursday, December 09, 2010 1:26 PM<BR><B>To:</B> 'Roy
Hantgan'; rasmb@rasmb.bbri.org<BR><B>Subject:</B> Re: [RASMB] radial scans 6
sector cells<BR></FONT><BR></DIV>
<DIV></DIV>
<DIV class=WordSection1>
<P class=MsoNormal><SPAN style="COLOR: #1f497d">Roy,<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="COLOR: #1f497d">Funny you should mention this, I
did my first 6-sector experiment yesterday. The 5.7 default might not cover the
inside sector, so you might have to adjust it to 5.65. I did this by trial and
error and by getting help from Dr. John Burgner. The 5.65 worked well I get
alittle of the top of the cell and both menisci. Hope this
helps.<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="COLOR: #1f497d">Lake<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="COLOR: #1f497d"><o:p> </o:p></SPAN></P>
<DIV>
<DIV
style="BORDER-BOTTOM: medium none; BORDER-LEFT: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 0in; PADDING-RIGHT: 0in; BORDER-TOP: #b5c4df 1pt solid; BORDER-RIGHT: medium none; PADDING-TOP: 3pt">
<P class=MsoNormal><B><SPAN
style="FONT-FAMILY: 'Tahoma','sans-serif'; FONT-SIZE: 10pt">From:</SPAN></B><SPAN
style="FONT-FAMILY: 'Tahoma','sans-serif'; FONT-SIZE: 10pt">
rasmb-bounces@rasmb.bbri.org [mailto:rasmb-bounces@rasmb.bbri.org] <B>On Behalf
Of </B>Roy Hantgan<BR><B>Sent:</B> Thursday, December 09, 2010 3:42
PM<BR><B>To:</B> rasmb@rasmb.bbri.org<BR><B>Subject:</B> [RASMB] radial scans 6
sector cells<o:p></o:p></SPAN></P></DIV></DIV>
<P class=MsoNormal><o:p> </o:p></P>
<P class=MsoNormal>Dear colleagues:<o:p></o:p></P>
<P class=MsoNormal><o:p> </o:p></P>
<P class=MsoNormal>Can you please remind me how you set the radial distance for
sed eq runs in 6-sector cells?<BR>I’ve only run sed eq in double sector cells.
<o:p></o:p></P>
<P class=MsoNormal><o:p> </o:p></P>
<P class=MsoNormal>Thanks for your advice,<o:p></o:p></P>
<P class=MsoNormal>Roy <o:p></o:p></P>
<P class=MsoNormal><o:p> </o:p></P>
<P class=MsoNormal><SPAN
style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 10pt">Roy R. Hantgan,
Ph.D.<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN
style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 10pt">Associate Professor
of Biochemistry / Associate in Molecular Medicine<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN
style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 10pt">Director,
Macromolecular Interactions Core Laboratory (MICL)<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN
style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 10pt">Wake Forest
University School of Medicine<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN
style="FONT-FAMILY: 'Arial','sans-serif'; FONT-SIZE: 10pt">3058 Hanes Bldg / TEL
336-716-4675<o:p></o:p></SPAN></P>
<P class=MsoNormal><o:p> </o:p></P>
<P class=MsoNormal><o:p> </o:p></P>
<P class=MsoNormal><o:p> </o:p></P></DIV></BODY></HTML>