[RASMB] Loading concentration

[Karl.Maluf at UCHSC.edu]

Borris (and anyone else),
 
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?
 
Thanks,
 
N. Karl Maluf
 
-----Original Message----- 
From: rasmb-admin at rasmb-email.bbri.org on behalf of Borries Demeler 
Sent: Tue 12/16/2003 7:33 PM 
To: Lin Fang 
Cc: rasmb at alpha.bbri.org 
Subject: Re: [RASMB] Loading concentration



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	>
	> Hi,
	>
	> I got my data for equilibrium analytical ultracentrifugation. It
	> turned out the absorbance of my protein is below 0.1 (OD280). Are
	> these data still analyzable? And what is the optimum range for
	> equilibrium analytical ultracentrifugation? Thanks a lot.
	>
	>
	> Lin
	
	Lin,
	
	you can probably shift the wavelength to 230 nm or lower to see if
	your protein absorbs better. You need to make sure that your buffer
	doesn't absorb at the lower wavelength. I generally include absorbance
	data up to 0.9 OD, give or take a few dependening on wavelength intensity.
	
	less than 0.1 OD sounds almost like nothing. At this OD 10% of the signal
	is noise which is too much to get reliable analysis for any system.
	
	It is best to use multiple concentration measurements and include all
	of them in your global fit. I generally use 2 6-channel cells with
	0.3, 0.5 and 0.7 OD 230 and 280. This gives me a broad concentration
	range over which I am analyzing the sample. For many cases this works
	very well. If you don't have aromatic sidechains in your protein, your
	mileage may vary at 280 nm.
	
	Good luck, -Borries
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