[RASMB] absorbance versus interference question

[Tom Laue]

Hi-
GST forms tight dimers, so finding two boundaries might correspond to 
dimer and incompetent monomer.
What are the sedimentation coefficients for the two boundaries and do 
they change with concentration?
Best wishes,
Tom

Leech, AP wrote:
> Hi Mark,
>
> You do not say how you are analysing the data. How are you producing the
> MW estimates?
>
> I would normally produce a sedimentation coefficient distribution first,
> e.g. SEDFIT's c(s). This should give very similar values for the peaks
> from the two sets of data. The s values are not so sensitive to the data
> quality as M values, which require an estimate of f/fo determined from
> the boundary shape. Boundary shape is much more susceptible to mis-
> interpretation.
>
> Another thing you can check is the signal amplitude - change between
> plateau at the experiment start and the baseline at the end. Using the
> extinction coefficient for the protein for the absorbance data, and the
> standard 3.3 fringes per mg/ml for the interference data will allow you
> to estimate how much material is sedimenting. If it's different for the
> two optics systems, you aren't observing the same material. (You can
> more conveniently use the integrations in SEDFIT for this. Also they
> will allow you to look at the relative amounts under the two peaks - are
> they the same?)
>
> All the best,
>
> Andrew
>
>
>
> Mark Agacan wrote:
>> Hello,
>>
>> i'm studying a ~ 45 kDa protein attached via a fixed-length linker to 
>> a GST tag (~ 30 kDa)
>>
>> SV 280nm-absorbance with quartz windows shows two separate peaks 
>> only, with corresponding mass of 30 and 45 kDa.
>>
>> SV interference scans collected during the same experiment, and with 
>> the quartz windows, shows two peaks with mass 75 and 150 kDa.
>>
>> I think the protein should sediment as a single protein-GST complex, 
>> so interference seems more reasonable.
>>
>> Gel shows the 75 and 150 kDa species but not the individual protein 
>> and GST-tag.  The
>> Why would absorbance pick up the protein-gst complex as two distinct 
>> species, while interference  finds the intact protein-GST monomer and 
>> dimer?
>>
>> In general I've used absorbance data at 280 nm with quarz windows to 
>> generate the continuous sedimentation distribution unless there are 
>> issues with absorbance at this wavelength.  However after this 
>> experiment I may change to using interference with sapphire windows 
>> as it seems to be more consistent with other data, i.e. gels, activity.
>>
>> Any ideas on this would be very welcome.
>>
>> Cheers
>>
>> Mark
>>
>>
>>
>> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
>> Dr Mark Agacan, Scientific Officer for the Division of Biological 
>> Chemistry and Drug Discovery,
>> Wellcome Trust Biocentre, College of Life Sciences, University of 
>> Dundee, Dundee, DD1 5EH Tel: +44 1382 386095    Fax: +44 1382 345764
>> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
>> The University of Dundee is a registered Scottish charity, No: SC015096
>> _______________________________________________
>> RASMB mailing list
>> RASMB at rasmb.bbri.org
>> http://rasmb.bbri.org/mailman/listinfo/rasmb
>

-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
Durham, NH 03824-3544
Phone: 603-862-2459
FAX:   603-862-0031
E-mail: Tom.Laue at unh.edu
www.bitc.unh.edu
www.camis.unh.edu