[RASMB] absorbance versus interference question

[Mark Agacan]

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



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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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