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--></style><title>Re: [RASMB] Can DCDT+ be used to detect small
change i</title></head><body>
<div>Hi Chris,</div>
<div><br></div>
<div><x-tab> </x-tab>Yes,
DCDT can be used to detect very small changes in s. If you match the
menisi using a capillary type synthetic boundary cell so the the
sample on each side starts sedimenting at the same position. You can
iether compare two samples directlyIf you are adding a low molecular
weight ligand, you must put the sample plus ligand on the
"sample" side , fill the "reference" side slightly
more (20uL) than the sample side with sample without ligand. Take the
run up to 3-10K rpm to match the menisci. Then stop the run, gently
shake the rotor to obtain a uniform distribution and restart. A small
amount of sample containing no ligand (10uL) will flow in to the
sample and cause a very small dilution of ligand. The sample must have
been dialyzed vs. the basic buffer without ligand before being
divided. When you add ligand to the sample also add an equal amount of
buffer to sample not containing buffer to dilute it by the same
amount. The ligand should be dissolved in the dialysate. Am assuming
that you are using the interference optics. The data can then be
analyzed with DCDT. by fitting the boundary to two gaussians - on
positive and one negative. I will give all the details if you contact
me directly. You can also fit the data using numerical solutions to
the Lamm equation using SedAnal.</div>
<div><br></div>
<div>Walter Stafford</div>
<div><br></div>
<div>At 13:55 -0500 9/20/02, Chin, Christopher wrote:</div>
<blockquote type="cite" cite><font size="-1">To John, all DCDT+
users and all interested colleagues,</font></blockquote>
<blockquote type="cite" cite><font
size="-1"> </font></blockquote>
<blockquote type="cite" cite><font size="-1"> John, I would like
to have your comment in the following three experiments using RNAseA
as a model and DCDT+ as the method to do data
analysis.</font></blockquote>
<blockquote type="cite" cite><font
size="-1"> </font></blockquote>
<blockquote type="cite" cite><font size="-1">Experiment
1: The purpose is to check the stability of RNAseA. The
comparison is made between the protein left at room temperature
for a couple of days and that storage in
the freezer.</font></blockquote>
<blockquote type="cite" cite><font size="-1">Experiment
2: Question: Can the denatured RNaseA (has 4 s-s bond)
in GuHCl be refolded back to its native state in the
absence of reducing agent?</font></blockquote>
<blockquote type="cite" cite><font size="-1">Experiment 3:
Question: Can the denatured RNaseA (has 4 s-s bond) in
GuHCl be refolded back to its native state in the presence of
reducing agent TBP (Tri-n-butylphosphine).</font></blockquote>
<blockquote type="cite" cite><font
size="-1"> </font></blockquote>
<blockquote type="cite" cite><font size="-1">The major issue here is
how I can differentiate the<i><b> DS</b></i> difference
due to scan chosen from the <i><b>DS</b></i> difference
due to real experimental finding (between the control and the
sample).</font></blockquote>
<blockquote type="cite" cite><font
size="-1"> </font></blockquote>
<blockquote type="cite" cite><font size="-1">I am pleased to
say that, using the criteria that I have stated, e.g. maximized
or magnified the<font face="Arial"><i><b> DS</b></i> difference due to
scan chosen </font>(use a smaller number for denominator when
converted to %), <font face="Arial">m</font>inimized or play
down the<font face="Times New Roman"><i><b> DS</b></i> difference
between the control and the sample (use a larger number for
denominator when converted to % </font></font><font
face="Times New Roman">),</font><font size="-1"> the results
are pretty much I have expected. Do you think
I try to push the instrument limit too far to obtain these
results by manipulating the treatment of
data?</font></blockquote>
<blockquote type="cite" cite><font
size="-1"> </font></blockquote>
<blockquote type="cite" cite><font size="-1">s20,w values reported
here are the results of fitting dcdt.</font></blockquote>
<blockquote type="cite" cite><font
size="-1"> </font></blockquote>
<blockquote type="cite" cite><font size="-1">Thank you in advance for
your comment or advice.</font></blockquote>
<blockquote type="cite" cite><font
size="-1"> </font></blockquote>
<blockquote type="cite" cite><font size="-1">Chris</font></blockquote>
<blockquote type="cite" cite><font size="-1"> </font><br>
</blockquote>
<blockquote type="cite" cite><font
size="-1"
>--------------------------------------------------------------------<span
></span>------<br>
Christopher Chin<br>
Manager, XLA-Analytical Ultracentrifugation facility<br>
Sealy Center for Structural Biology<br>
HBC&G, 5.134 MRB.UTMB, Galveston,TX 77555-1055<br>
cchin@utmb.edu, 409-772-1693, efax 708-585-1920<br>
---------------------------------------------------------------------<span
></span>------</font><br>
</blockquote>
<blockquote type="cite" cite><font size="-1"><br></font></blockquote>
<blockquote type="cite" cite><br>
Attachment converted: WFS3HD:Stability of RNAse.doc (WDBN/MSWD)
(00121490)<br>
Attachment converted: WFS3HD:GuHCl treated(without reducing
(WDBN/MSWD) (00121491)<br>
Attachment converted: WFS3HD:GuHCl-TBP treated and renatural
(WDBN/MSWD) (00121492)</blockquote>
<div><br></div>
<div>-- <br>
#####################################################################<span
></span>###<br>
<br>
Walter F. Stafford III, Ph.D.<br>
Senior Scientist<br>
Analytical Ultracentrifugation Research Laboratory<br>
Boston Biomedical Research Institute<br>
64 Grove Street<br>
Watertown, MA 02472-2829<br>
and<br>
Associate in Neurology<br>
Massachusetts General Hospital<br>
Harvard Medical School<br>
Boston MA 02115<br>
<br>
main:(617) 926-8040<br>
tel: (617) 658-7808<br>
fax: (617) 972-1753<br>
<br>
mailto:stafford@bbri.org<br>
<br>
<br>
http://www.bbri.org/faculty/stafford/Stafford.html<br>
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