[RASMB] Determining meniscus in fluorescence scans using Aviv FDS

[John J. Correia]

rishi

We find is useful to scan the cells after the FDS run in ABS mode at the same speed.  It helps determine when rm really is.  Another approach in more complex model analysis is to fix the s value of a known species and fit for rm - we do this in sedanal.  Once you have rm the rest of the analysis can proceed.


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Please note my email has changed to jcorreia at umc.edu

Dr. John J. "Jack" Correia
Department of Biochemistry
University of Mississippi Medical Center
2500 North State Street
Jackson, MS  39216
(601) 984-1522
fax (601) 984-1501
email address: jcorreia at umc.edu
homepage location: http://biochemistry.umc.edu/correia.html
dept homepage location:    http://biochemistry.umc.edu/
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From: rasmb-bounces at rasmb.bbri.org [rasmb-bounces at rasmb.bbri.org] On Behalf Of rjsanyal at u.washington.edu [rjsanyal at u.washington.edu]
Sent: Friday, February 25, 2011 2:06 AM
To: RASMB at server1.bbri.org
Subject: [RASMB] Determining meniscus in fluorescence scans using Aviv FDS

Dear RASMB community,

I'm using the Aviv fluorescence detection system (FDS) installed in a Beckman XL-A/XL-I ultracentrifuge here at the University of Washington. I'm studying protein-DNA interactions between a viral motor protein & a fluorescently-labeled dsDNA substrate containing the wild-type viral binding sequence of the protein.

My question concerns the placement of the meniscus indicator in the fluorescence data I obtain. Specifically, when using Sedfit. For absorbance data, the meniscus is much more clear & can be automatically found by Sedfit if you have reasonable boundaries set for the fit. When doing the same using Sedfit for fluorescence data, Sedfit simply seems to search for the local max within the window defined.

A cursory search on RASMB did not turn up any info, but I do apologize if this has already been covered before. My concern stems mainly from my wanting to set the meniscus properly to obtain accurate S values, & also from the fact that I've noticed that in my own hands even when I try to set the meniscus to some characteristic feature of the data sets (say, the beginning of the first scan), I do see a variation of 0.2-0.5S for some arbitrary resulting species when using the same data set analyzed on different occasions. I'd assume that's beyond the sort of tolerance for meniscus placement I should be looking for, especially when protein binding itself can, minimally in our system, lead to a 0.5S shift.

Many thanks in advance for your help,
Rishi

~~~~~~~~~~~~~~~
Rishi Sanyal |Graduate Student
Catalano Lab | Univ. of Washington, Seattle WA
rjsanyal at uw.edu

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