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<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=170505516-04062002>Ruby,</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=170505516-04062002></SPAN></FONT> </DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN class=170505516-04062002>I
doubt your problems have to do with non-ideality. I assume you meant a
charge of -1.5, not a pI. You never said what protein concentrations you are
using, and salt can never get rid of the excluded volume portion of nonideality,
but I certainly would expect that .25 M of NaCl would suppress the charge
repulsion.</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=170505516-04062002></SPAN></FONT> </DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN class=170505516-04062002>My
guess is that your problems are related to the DTT, not nonideality. You will
have variable amounts of DTT oxidation occurring over the course of the run,
causing shifting baseline offsets. See earlier discussions on RASMB about DTT
and other reductants.</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=170505516-04062002></SPAN></FONT> </DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN class=170505516-04062002>[To
the group] Speaking of reductants, I noticed recently that Pierce is selling
TCEP immobilized on gel particles. I'm wondering if these could be put into the
cell and basically keep the reductant down at the cell base and (mostly) out of
the light beam. Has anyone tried this? The literature I saw didn't indicate
the particle size, so I don't know whether they would fit through the
loading holes in a 2-channel or external loading 6-channel centerpiece, but
presumably this would at least be possible in a regular
6-channel.</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=170505516-04062002></SPAN></FONT> </DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN class=170505516-04062002>John
Philo</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=170505516-04062002>Alliance Protein Labs</SPAN></FONT></DIV>
<BLOCKQUOTE dir=ltr style="MARGIN-RIGHT: 0px">
<DIV class=OutlookMessageHeader dir=ltr align=left><FONT face=Tahoma
size=2>-----Original Message-----<BR><B>From:</B>
rasmb-admin@rasmb-email.bbri.org
[mailto:rasmb-admin@rasmb-email.bbri.org]<B>On Behalf Of </B>Yun-Ru (Ruby)
Chen<BR><B>Sent:</B> Tuesday, June 04, 2002 9:03 AM<BR><B>To:</B>
rasmb@rasmb-email.bbri.org<BR><B>Subject:</B> [RASMB] Fw: Can 0.25M of NaCl
take care of nonideal behavior of a protein?<BR><BR></FONT></DIV>
<DIV><FONT face=Arial size=2></FONT> </DIV>
<DIV style="FONT: 10pt arial">----- Original Message -----
<DIV style="BACKGROUND: #e4e4e4; font-color: black"><B>From:</B> <A
title=ychen5@unity.ncsu.edu href="mailto:ychen5@unity.ncsu.edu">Yun-Ru (Ruby)
Chen</A> </DIV>
<DIV><B>To:</B> <A title=rasmb@rasmb-email.bbri.org
href="mailto:rasmb@rasmb-email.bbri.org">rasmb@rasmb-email.bbri.org</A> </DIV>
<DIV><B>Sent:</B> Friday, May 31, 2002 2:37 PM</DIV>
<DIV><B>Subject:</B> Can 0.25M of NaCl take care of nonideal behavior of a
protein? </DIV></DIV>
<DIV><BR></DIV>
<DIV><FONT face=Arial size=2>Dear RASMB members:</FONT></DIV>
<DIV><FONT face=Arial
size=2>
I have currently run into problems by analyzing my data from
sedimentation equilibrium studies. I am very confused and frustrated
about it.</FONT></DIV>
<DIV><FONT face=Arial size=2>I hope if any of you can give me some
ideas. I am working on a 10KDa protein domain which is suppose to be a
helical bundle. The pI of the protein is calculated to be about -1.5 at pH 8.
I have ran the samples in two different buffers, one in 30 mM Tris, pH8, and
one in Tris with 250 mM NaCl. Both buffers contain 0.2mM DTT and are
at pH 8. However, we cannot fit all the data to a single assembly
model. I have tried to put in different guesses of "B" values for
nonideality, but the fits are even worse. Moreover, the data
from the salt buffers look better then the one without. Therefore, does
that mean 0.25M NaCl is not enough or there is something else that I
should be aware of? My protein is much less soluble in the presence
of NaCl, so it is a bit hard for me to increase to salt
concentrations.</FONT></DIV>
<DIV><FONT face=Arial size=2>Thanks very much if you can share
your idea with me.</FONT></DIV>
<DIV><FONT face=Arial size=2></FONT> </DIV>
<DIV><FONT face=Arial size=2>Sincerely,</FONT> </DIV>
<DIV><FONT face=Arial size=2>Ruby</FONT></DIV>
<DIV><FONT face=Arial size=2></FONT> </DIV>
<DIV><FONT face=Arial size=2>Yun-Ru (Ruby) Chen<BR>Ph.D.
candidate<BR>Department of Structural and Molecular Biochemistry<BR>North
Carolina State University</FONT></DIV></BLOCKQUOTE></BODY></HTML>