[RASMB] Determining meniscus in fluorescence scans using Aviv FDS

Cole, James james.cole at uconn.edu
Sat Feb 26 07:33:47 PST 2011


Dear Rishi
We have used the method described in the Perugini paper as well as scanning
with absorbance after the run as described by Jack and they give the same
values for the meniscus position. We were a bit concerned that some of the
lipophilic dye might bind to the protein in the sample, but we have not
found it to be a problem for the system we tested. I'd certainly do the
control for each system you look.

Good luck,
Jim Cole



On 2/25/11 4:18 PM, "John J. Correia" <jcorreia at umc.edu> wrote:

> 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.
> 
> 
> -------------------------------------------------------------------
> 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/
> -------------------------------------------------------------------
> ________________________________________
> 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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