[RASMB] Determining meniscus in fluorescence scans using Aviv FDS

[rjsanyal at u.washington.edu]

I'd just like to thank everyone for their input. This discussion has certainly given me a number of avenues to explore to get the best results with the FDS. What a great community.

Many thanks,
Rishi


On Sat, 26 Feb 2011, Cole, James wrote:

> 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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