[RASMB] fringe deviation

[John Philo]

Barbara,

What you are forgetting is that if you are reaching concentrations of 50
fringes (~16 mg/ml) then you are well into the range where non-ideality is
significant, and you really MUST treat these as non-ideal solutions.

For that same reason, and others, I cannot agree with Judith's suggestion.
Non-ideality effects are much stronger in velocity than equilibrium.
Further, it is doubtful that you could even do a velocity experiment at 16
mg/ml without exceeding the interference optics ability to track the steep
fringe gradient. A 3 mm pathlength cell would make the experiment feasible
but doesn't get around the non-ideality issue.

Lastly, while I'm certainly a great fan of sedimentation velocity, and yes
one can now treat self-associations in velocity, in my view you will get
much more robust results for the equilibrium constant from equilibrium data.
It is much easier, and usually quicker, to cover the broad range of
concentrations that is needed using equilibrium since you can easily do 9
concentrations at once using 6-channel cells (and monomer-dimer systems need
a VERY wide range of concentration). Further, the data analysis is more
robust for equilibrium because there are fewer fitting parameters (in
velocity you need to fit the sedimentation coefficient for each species
also).

John Philo
Alliance Protein Laboratories

-----Original Message-----
From: rasmb-admin at rasmb-email.bbri.org
[mailto:rasmb-admin at rasmb-email.bbri.org] On Behalf Of
judithk at vax2.concordia.ca
Sent: Monday, February 16, 2004 12:30 PM
To: lelj
Cc: rasmb at alpha.bbri.org
Subject: Re: [RASMB] fringe deviation


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Barbara, Why don't you continue with velocity experiments if you have
evidence from them for an equilibrium? By doing velocity experiments at
different concentrations of proteins, you should be able to determine Kd. P.
Schuck's program - SEDPHAT - might be useful for the data analysis.

Judith Kornblatt
Dept of Chemistry and Biochemistry
Concordia University
7141 Sherbrooke Ouest
Montreal, Qc H4B 1R6
Tel: 1 514 848 2424, ext 3384   FAX: 1 514 848 2868




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