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<DIV><SPAN class=890434514-11042002><FONT face=Arial color=#0000ff size=2>Pascal
and RASMB,</FONT></SPAN></DIV>
<DIV><SPAN class=890434514-11042002><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=890434514-11042002><FONT face=Arial color=#0000ff
size=2>Overall I think there is perhaps too much concern about the issue of scan
speed for absorbance scans with respect to DCDT analysis, which in the extreme
has led some people to believe this approach can't be used at all (definitely
false!). It is important to remember that the primary determinant of
signal/noise in the DCDT method is the magnitude of the difference between
the first and last scans used in the analysis, and that is really determined by
the masses of the species in the sample (the time span must be limited to avoid
peak broadening) and that difference is <U>independent of the scan speed</U>.
Once the time span from first to last scan is fixed, the signal/noise only
increases as the square root of the number of scans, so even factors of 2 in
scan speed have fairly small impact on the overall
signal/noise.</FONT></SPAN></DIV>
<DIV><SPAN class=890434514-11042002><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=890434514-11042002><FONT face=Arial color=#0000ff size=2>I do
not recommend that you use replicates in velocity scans, and especially not in
the 'continuous' scan mode (as opposed to 'step' scan). In continuous scan mode
the slit is still moving as it takes the replicates, and thus the readings from
different radii are getting averaged together (a bad idea). Further, in my view
a spacing of .02 cm is far too large. For one thing, that large spacing means
the subtraction of time-invariant noise from scratches or dirt on the windows
(one of the primary advantages of the DCDT approach) does not work well
because the radial profiles are so sparse.</FONT></SPAN></DIV>
<DIV><SPAN class=890434514-11042002><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=890434514-11042002><FONT face=Arial color=#0000ff size=2>I'm
not sure why you saw a significant difference between 2 versus 3 replicates; in
theory this should make little difference, assuming you increased the number of
scans used in the analysis for the 2-replicate data, since the scan speed was
faster. (Remember to use the 'Test time range for broadening' function in DCDT+
to help you decide how many scans to use.)</FONT></SPAN></DIV>
<DIV><SPAN class=890434514-11042002><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=890434514-11042002><FONT face=Arial color=#0000ff size=2>My
recommendation is that you use the default settings for velocity runs:
continuous mode, .003 cm spacing, 1 replicate. To optimize things, you do want
to reset the default radial range (5.8 to 7.3 cm) so you scan from just to the
left of the meniscus down to ~7.10-7.14 cm, avoiding the region at the cell base
were solutes accumulate (<EM>i.e. </EM>don't waste time taking data you can't
use in the analysis). That will give a scan rate of about 80-90 seconds per
cell.</FONT></SPAN></DIV>
<DIV><SPAN class=890434514-11042002><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=890434514-11042002><FONT face=Arial color=#0000ff
size=2>Regarding rotor speed, the faster you spin the better the resolution
among different species, but you must balance this against loss of signal/noise
because you have fewer scans to analyze. Also for higher mass species at some
point high speed gives boundaries so sharp the diffusion coefficient (and hence
mass) cannot be determined accurately. In your case I would think 50-55 K is
about right.</FONT></SPAN></DIV>
<DIV><SPAN class=890434514-11042002><FONT face=Arial color=#0000ff
size=2></FONT></SPAN> </DIV>
<DIV><SPAN class=890434514-11042002><FONT face=Arial color=#0000ff size=2>John
Philo</FONT></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>pascal
egea<BR><B>Sent:</B> Wednesday, April 10, 2002 5:53 PM<BR><B>To:</B>
rasmb@rasmb-email.bbri.org<BR><B>Subject:</B> [RASMB] velocity
sedimentation<BR><BR></FONT></DIV>
<DIV><FONT face=Arial size=2>Greetings,</FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2></FONT> </DIV>
<DIV><FONT face=Arial size=2>I am currently doing some velocity sedimentation
experiments using absorbance optics on a 33 kDal protein and I am processing
my data with DC/DT plus.</FONT></DIV>
<DIV><FONT face=Arial size=2>I have several questions to ask to the g(S)
function specialist.</FONT></DIV>
<DIV><FONT face=Arial size=2>first I was spinning at 50.000 rpm and scanning 3
replicates with a radial increment of 0.02 this was resulting in a scan every
3:30 minutes (roughly).</FONT></DIV>
<DIV><FONT face=Arial size=2>my g(S) function is very nice clearly indicating
a single component system with a S of about 2.3 S.</FONT></DIV>
<DIV><FONT face=Arial size=2>As It seems that the more frequently you scan the
better it is, I therefore tried only 2 replicates and still a radial increment
of 0.02 and now it allows me to scan every 2 minutes (approximately).
nevertheless my function does not look as nice (this is the same sample). I
also increased the speed to 55.000 rpm.</FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2></FONT> </DIV>
<DIV><FONT face=Arial size=2>so is it a result of a lower number of replicates
(more noise)?. Is it best to scan more frequently with more
noise versus less frequently with more replicates?</FONT></DIV>
<DIV><FONT face=Arial size=2>Is there for sedimentation velocity a really
optimal speed (I know the reference curve) or the faster you spin the better
it is?</FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2></FONT> </DIV>
<DIV><FONT face=Arial size=2>Thanks in advance for your answers.</FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2></FONT> </DIV>
<DIV><FONT face=Arial size=2>Pascal Egea</FONT></DIV>
<DIV><FONT face=Arial size=2>UCSF department of Biophysics and
Biochemistry</FONT></DIV></BLOCKQUOTE></BODY></HTML>