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<TITLE>Re: [RASMB] Loading concentration</TITLE>
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<DIV>Borris (and anyone else),</DIV>
<DIV> </DIV>
<DIV>For a global analysis of sedimentation equilibrium data obtained over a
range of wavelengths, do you use extinction coefficients calculated using the
XLA absorbance optics, or do you use values obtained with more precise
insturmentation? Often, when one sets a scan at 230nm, the value written
to the data file is off +/- 1nm. At 230nm, this can change the absorbance
by a significant amount. Which value is correct - the one entered or the
one written to the data file? Finally, you indicated you don't trust data
below 0.1 OD. If you have collected an absorbance scan that ranges from
below 0.1 to a value around, say, 0.9, do you exclude data below 0.1 in the
analysis? I agree one should not trust data if the entire scan is below
0.1 OD, but if the data spans a wide range of OD's, what is the most appropriate
data to include?</DIV>
<DIV> </DIV>
<DIV>Thanks,</DIV>
<DIV> </DIV>
<DIV>N. Karl Maluf</DIV>
<DIV> </DIV>
<DIV><FONT size=2>-----Original Message----- <BR><B>From:</B>
rasmb-admin@rasmb-email.bbri.org on behalf of Borries Demeler
<BR><B>Sent:</B> Tue 12/16/2003 7:33 PM <BR><B>To:</B> Lin Fang <BR><B>Cc:</B>
rasmb@alpha.bbri.org <BR><B>Subject:</B> Re: [RASMB] Loading
concentration<BR><BR></DIV></FONT>
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Hi,<BR>><BR>> I got my data for equilibrium analytical
ultracentrifugation. It<BR>> turned out the absorbance of my protein is
below 0.1 (OD280). Are<BR>> these data still analyzable? And what is the
optimum range for<BR>> equilibrium analytical ultracentrifugation? Thanks a
lot.<BR>><BR>><BR>> Lin<BR><BR>Lin,<BR><BR>you can probably shift the
wavelength to 230 nm or lower to see if<BR>your protein absorbs better. You
need to make sure that your buffer<BR>doesn't absorb at the lower wavelength.
I generally include absorbance<BR>data up to 0.9 OD, give or take a few
dependening on wavelength intensity.<BR><BR>less than 0.1 OD sounds almost
like nothing. At this OD 10% of the signal<BR>is noise which is too much to
get reliable analysis for any system.<BR><BR>It is best to use multiple
concentration measurements and include all<BR>of them in your global fit. I
generally use 2 6-channel cells with<BR>0.3, 0.5 and 0.7 OD 230 and 280. This
gives me a broad concentration<BR>range over which I am analyzing the sample.
For many cases this works<BR>very well. If you don't have aromatic sidechains
in your protein, your<BR>mileage may vary at 280 nm.<BR><BR>Good luck,
-Borries<BR>_______________________________________________<BR>RASMB mailing
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