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<DIV><FONT face=Arial size=2>Hi, Dr. Laue:</FONT></DIV>
<DIV><FONT face=Arial
size=2> Thank you for your
kindly reply. I have sent out an email describing my conditions,
but I don't think it has been posted. Here are the information that I
provided in my last email.</FONT></DIV>
<DIV> "The concentrations of my sample
are as follows: For the ones in Tris/DTT buffer without salt, they are 121, 60,
and 30 uM (Abs @280=1, 0.5,<BR>0.25). For those in 0.25M NaCl/ Tris/DTT buffer,
they are 79, 36, and 18 uM.(Abs at 280 = 0.65, 0.3 , 0.15). I have rechecked my
samples by SDS-PAGE,<BR>they looked fine, at least no other major band present.
The speeds I used are 24, 32, and 45K rpm. The sigma factors for those speeds in
order are 0.7433,<BR>1.3215, and 2.613. The sigma factor is calculated by the
definition (Mb*w^2)/RT.<BR> About the DTT
problems, I have 0.2mM DTT in my samples. The thing is that my protein has only
one Cysteine. Therefore, there is no<BR>intramolecular S-S bonds, but perhaps a
potential of forming intermolecular S-S
bonds."<BR> The B often comes out negative.
Recently, I have some more progress. I have pretty good results from gel
exclusion showing this protein is a 1-4-8 (even 10)</DIV>
<DIV>association. I rechecked my AUC SE data and found I can fit them all to a
1-4-8 model. The octamer is much less than tetramer, also the tetramer is much
less than octamer. When I replace DTT (1 mM) by TCEP
(0.5mM) in my buffer (250mM NaCl, 30mM Tris, pH 8), I don't see
the octamer anymore. But I still have to confirm this since the loading sample
concentration is about 1/3 different. </DIV>
<DIV> I will check the v bar carefully. We
would like to do the sedimentation equilibrium again once we have a better idea
from the sizing column. Does anyone know what TCEP concentrations is
good for AUC studies? And should I do sedimentation velocity to get more
information?</DIV>
<DIV><FONT face=Arial size=2> Dr. Laue, I am
very happy to get your reply since I have given a student seminar on AUC and
have heard and read about you.</FONT></DIV>
<DIV><FONT face=Arial size=2>Regards,</FONT></DIV>
<DIV><FONT face=Arial size=2>Ruby</FONT></DIV>
<DIV>----- Original Message ----- </DIV>
<BLOCKQUOTE dir=ltr
style="PADDING-RIGHT: 0px; PADDING-LEFT: 5px; MARGIN-LEFT: 5px; BORDER-LEFT: #000000 2px solid; MARGIN-RIGHT: 0px">
<DIV
style="BACKGROUND: #e4e4e4; FONT: 10pt arial; font-color: black"><B>From:</B>
<A title=Tom.Laue@unh.edu href="mailto:Tom.Laue@unh.edu">Tom Laue</A> </DIV>
<DIV style="FONT: 10pt arial"><B>To:</B> <A title=ychen5@unity.ncsu.edu
href="mailto:ychen5@unity.ncsu.edu">Yun-Ru (Ruby) Chen</A> </DIV>
<DIV style="FONT: 10pt arial"><B>Sent:</B> Friday, June 14, 2002 10:07
AM</DIV>
<DIV style="FONT: 10pt arial"><B>Subject:</B> RE: [RASMB] Fw: Can 0.25M of
NaCl take care of nonideal behavior of a protein?</DIV>
<DIV><FONT face=Arial size=2></FONT><FONT face=Arial size=2></FONT><BR></DIV>
<DIV><SPAN class=532175213-14062002><FONT face=Arial color=#0000ff size=2>Hi
Ruby,</FONT></SPAN></DIV>
<DIV><SPAN class=532175213-14062002><FONT face=Arial color=#0000ff
size=2>Sorry for the tardy reply. I looked over the responses you got and
agree with John Philo that it is most likely that your problems stem from
baseline offset. A couple of questions: Are you using absorbance optics? At
what wavelengths? What concenrations of protein are you using? What rotor
speeds did you use?</FONT></SPAN></DIV>
<DIV><SPAN class=532175213-14062002><FONT face=Arial color=#0000ff size=2>When
you fit, does B come out negative or positive? If it is negative, then you are
looking at association/aggregation. If B is positive, then nonideality is the
culprit. The charge on the peptide is not extreme, so I sincerely doubt that
nonideality is a problem in either solvent. If you do not get the expected
molecular weight when you analyze the data, it may be because of the vbar
calculation. In particular, if the peptide has a number of charged residues,
the calculated vbar may be to large, leading to estimates of M that are too
large. This will be the case even if the net charge is small (similar to what
you describe). My experience is that the vbar is about 0.02-0.03 lower if
there are ~10-15 charged residues/peptide of this size.</FONT></SPAN></DIV>
<DIV><SPAN class=532175213-14062002><FONT face=Arial color=#0000ff size=2>Best
wishes,</FONT></SPAN></DIV>
<DIV><SPAN class=532175213-14062002><FONT face=Arial color=#0000ff size=2>Tom
Laue</FONT></SPAN></DIV>
<DIV><SPAN class=532175213-14062002><!-- Converted from text/rtf format -->
<P><SPAN lang=en-us><FONT face=Arial size=2>University of New
Hampshire</FONT></SPAN> <BR><SPAN lang=en-us><FONT face=Arial size=2>46
College Road</FONT></SPAN> <BR><SPAN lang=en-us><FONT face=Arial size=2>Rudman
379</FONT></SPAN> <BR><SPAN lang=en-us><FONT face=Arial size=2>Durham, NH
03824</FONT></SPAN> <BR><SPAN lang=en-us><FONT face=Arial size=2>Phone:
603-862-2459</FONT></SPAN> <BR><SPAN lang=en-us><FONT face=Arial
size=2>FAX: 603-862-0031</FONT></SPAN> <BR><SPAN
lang=en-us><FONT face=Arial size=2>www.camis.unh.edu</FONT></SPAN> <BR><SPAN
lang=en-us><FONT face=Arial size=2>www.bitc.unh.edu</FONT></SPAN>
</P></SPAN></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 12:03 PM<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></BLOCKQUOTE></BODY></HTML>