[RASMB] absorbance versus interference question

[Arthur Rowe]

Hi Mark

Is this a one-off, or has the experiment been repeated? On the face of  
it, what you report is not possible, assuming the laws of physics have  
not been suspended. Is there any clue in your fits (SEDFIT?) - e.g. are  
the estimates for the meniscus position in absorption/interference  
consistent? {occasionally one gets a fit wandering of into wild  
country} Do the estimates for the frictional ratio make sense, assuming  
your protein ±GST tag are more or less 'globular'?

With a complex and its components having different extinction  
coefficients there is quite a bit to be said for using interference  
optics, for which the dn/dc value is more or less invariable, at least  
at usual ionic strength & pH. But you do need to sort out why the  
absorption optics are giving weird data - there will be plenty of times  
you will need them.

Kind regards

Arthur

************************************************************************ 
*******
Arthur J Rowe
Professor of Biomolecular Technology / Director NCMH Business Centre
School of Biosciences
University of Nottingham
Sutton Bonington
Leics LE12 5RD

TEL:  0115 9516156
FAX:  0115 0516157
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On Sep 29, 2009, at 17:07, 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
> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
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