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<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff><SPAN
class=881580009-03082011>Hi Titus,</SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff><SPAN
class=881580009-03082011></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff><SPAN
class=881580009-03082011>what is the density of a 100 mg/mL Ficoll 70 solution,
compared to your protein? Can you match the density of the ficoll-solvent to the
inverse density of the protein (if need be by addition of D2O, etc)? If so, you
could perform a density equilibrium experiment and use the width of the protein
equilibrium band as an indicator of the molar mass/diffusion coefficient (cf.
Baldwin PNAS 45:939; there is also more detailed work on the behaviour of
proteins and the relationship to the diffusion coefficient in
CsCl2-gradients in the 70s, but I'm still looking for that name). Or you
could try to measure the diffusion coefficient in an artificial boundary
experiment, that would save you the problem of the ficoll-gradients at high
speeds).</SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff><SPAN
class=881580009-03082011></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff><SPAN
class=881580009-03082011>Are you sure that the apparent stabilising effect
you observe is specific to Ficoll and not unspecifically caused by molecular
crowding?</SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff><SPAN
class=881580009-03082011></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff><SPAN
class=881580009-03082011>Best, Holger</SPAN></FONT></DIV><BR>
<DIV class=OutlookMessageHeader lang=en-us dir=ltr align=left>
<HR tabIndex=-1>
<FONT face=Tahoma><B>From:</B> rasmb-bounces@rasmb.bbri.org
[mailto:rasmb-bounces@rasmb.bbri.org] <B>On Behalf Of </B>Titus M.
Franzmann<BR><B>Sent:</B> Tuesday, August 02, 2011 8:32 PM<BR><B>To:</B> Arthur
Rowe<BR><B>Cc:</B> rasmb@rasmb.bbri.org<BR><B>Subject:</B> Re: [RASMB] AUC in
the presence of 100 mg/ml Ficoll70<BR></FONT><BR></DIV>
<DIV></DIV>
<DIV dir=ltr><FONT class=Apple-style-span face=Tahoma>Hi Arthur, </FONT>
<DIV style="FONT-SIZE: 10pt; FONT-FAMILY: Tahoma">thanks for your
reply. </DIV>
<DIV><FONT class=Apple-style-span face=Tahoma>From SV experiment in buffer we
learned that we thus far deal with a ~600 kd oligomer, however the protein only
weakly associates and forms a heterogeneous solution. From concentration
dependent biochemical activity experiments in buffer and buffer supplemented
with ficoll we assume</FONT><SPAN class=Apple-style-span
style="FONT-SIZE: 10pt; FONT-FAMILY: Tahoma"> that Ficoll stabilizes an
active oligomeric species. We wanted to directly confirm that this effect
is related to the formation of a greater and more homogeneous steady-state
population of possibly 600 kD oligomers.</SPAN></DIV>
<DIV>Best</DIV>
<DIV>Titus<BR><BR><FONT class=Apple-style-span face=Tahoma>Titus M.
Franzmann</FONT><BR><FONT class=Apple-style-span face=Tahoma>S. G. Walter
Lab</FONT><BR><FONT class=Apple-style-span face=Tahoma>Mol., Cell. and Develop.
Biol. Dept.</FONT><BR><FONT class=Apple-style-span face=Tahoma>University of
Michigan</FONT><BR><FONT class=Apple-style-span face=Tahoma>4140C Nat. Sci.
Bldg</FONT><BR><FONT class=Apple-style-span face=Tahoma>830 N. University
Ave.</FONT><BR><FONT class=Apple-style-span face=Tahoma>Ann Arbor, MI
48109-1048</FONT><BR><FONT class=Apple-style-span
face=Tahoma>titusfranzmann.wordpress.om</FONT><BR><BR>
<DIV style="FONT-SIZE: 10pt; FONT-FAMILY: Tahoma">
<HR id=stopSpelling>
CC: rasmb@rasmb.bbri.org<BR>From: arthur.rowe@nottingham.ac.uk<BR>Subject: Re:
[RASMB] AUC in the presence of 100 mg/ml Ficoll70<BR>Date: Tue, 2 Aug 2011
18:57:31 +0100<BR>To: tmfr@umich.edu<BR><BR>
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<FONT color=maroon>Hi Titus<BR><BR>The first thing that occurs to me is "how big
is your protein/whatever, and what are you trying to find out? Also - have you
tried simply putting your system into 100 mg/ml Ficol? I ask because although it
is advertised (I am sure correctly) as having "low toxicity", my own experience
(many years ago) with trying to fractionate muscle filaments in Ficol was that
this solvent was not exactly benign towards my precious
samples!<BR><BR>Obviously in SV the Ficol 70 will sediment, and although it will
be invisible (give or take a bit of turbidity) in the u/v, you will get some
very weird Johnston-Ogston effects if the s values of your system coincide at
all closely with those of the Ficol. Even if your reference channel contained
the 100 mg/ml Ficol, you could still have problems. SE sounds at least as
problematic - and although you would not be plagued by things such as
Johnston-Ogston effects, non-ideality/crowding effects sound
daunting.<BR><BR>Kind regards<BR><BR>Arthur<BR></FONT><BR><BR>On Aug 2, 2011, at
14:26, Titus M. Franzmann wrote:<BR><BR>
<DIV class=ecxEmailQuote><FONT size=+1>Hi,</FONT></DIV>
<DIV class=ecxEmailQuote><FONT size=+1>I was wondering if anyone could comment
on whether there is a (proper) way to perform either AUC SV (preferred) or EQ
with 100 mg/ml Ficoll70 in the buffer and which would be the best way to analyze
such data.</FONT></DIV>
<DIV class=ecxEmailQuote><FONT size=+1>Thanks for your assistance.</FONT></DIV>
<DIV class=ecxEmailQuote><FONT size=+1>Titus</FONT></DIV>
<DIV
class=ecxEmailQuote>_______________________________________________<BR>RASMB
mailing list<BR>RASMB@rasmb.bbri.org<BR><A
href="http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb"
target=_blank>http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb</A><BR><BR></DIV>*******************************************************************************<BR>Arthur
J Rowe<BR>Professor of Biomolecular Technology / Director NCMH Business
Centre<BR>School of Biosciences<BR>University of Nottingham<BR>Sutton
Bonington<BR>Leics LE12 5RD<BR><BR>TEL: 0115
9516156<BR><BR>*******************************************************************************<BR></DIV></DIV></DIV></BODY></HTML>