[RASMB] Weak association?

[John Correia]

Andrew
 
It would be helpful to provide the salt conditions, what is the ionic strength, and the calculated charge at the pH of your runs.  This would put the discussion of nonideality masking association in a real context.  You have done experiments up to ~71 uM so one can guess the Kd might be at least 710 uM and above since 10% association should easily be visible in a velocity run.  In practice affinities of 10^4 M-1 are on the edge of measurable.  The suggestion to fit the data to nonassociating vs dimer vs nonideal dimer is reasonable if you constrain the parameters in some reasonable way while comparing rms of the fits.  The nonideal dimer system is intrinsically difficult because nonideality and association strongly correlate.
 


>>> On 2/2/2009 at 8:22 AM, in message <49870199.3050700 at york.ac.uk>, "Leech, AP" <apl3 at york.ac.uk> wrote:
Hello all,

I have done some SV runs at various concentrations 0.3-10 mg/ml on
a protein (~14 ka) and the sedimentation coefficient appears to
remain constant (about 1.58). I had expected it to drop slightly at
higher concentrations, and so I suspect a weak self association.
Buffer is NaCl/Tris.

Is it reasonable to try and estimate an association coefficient
from this sort of data, and what is the best way to do it? (Even
a lower limit would be acceptable, as the intent is to show that
dimerisation is not significant).

I'm working my way through Gilbert & Gilbert at the moment (Meth.
Enzymol vol 27, p273) but is there perhaps something more in the
"global analysis" line?

All comments appreciated,

Andrew

-- 
Dr Andrew Leech                   *  Laboratory Manager
Technology Facility               *  Molecular Interactions Laboratory
Department of Biology (Area 15)   *  Tel   : +44 (0)1904 328723
University of York                *  Fax   : +44 (0)1904 328804
PO Box 373,  York  YO10 5YW       *  Email : apl3 at york.ac.uk 
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