[RASMB] absorbance versus interference question

[Leech, AP]

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 
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-- 
Dr Andrew Leech                   *  Laboratory Head
Technology Facility               *  Molecular Interactions Laboratory
Department of Biology (Area 15)   *  Tel   : +44 (0)1904 328723
University of York                *  Fax   : +44 (0)1904 328804
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