From: Rose Disher <disherr@centocor.com>
  To  : rasmb@alpha.bbri.org
  Date: Tue, 07 Sep 1999 09:31:01 -0400

Re: significance?

What data do you have from other methods which might enlighten you?  I
know I am taking the risk of stating the obvious, but everyone, myself
included, needs the occasional reminder of things we already know.  While
SDS-PAGE or size exclusion HPLC may not be helpful if the self-association
is reversible or occurs at high concentration, either technique  might
give you clues about the relative MW's if the association occurs at low
concentration and/or is irreversible.  If the structure and stability of
the molecule allow, a crosslinking reaction under mild conditions after
the aggregates have been formed might have the effect of making the
association irreversible.  There are even reagents available which would
allow you to cleave the crosslinking to return the protein to its
monomeric state.  If you are successful in freezing the association, you
might be able to get your answer from one of the above techniques or mass
spec.   I have had good luck with this sort of approach in the past,
although the reaction can be difficult to control if not carefully
planned.  I recommend Pierce as a good starting place for obtaining these
reagents.  Alternatively, you might try some other experimental techniques
to determine a value for one of the parameters you are currently floating
in one of the models and work via the process of elimination.

>>> Joel Mackay <j.mackay@biochem.usyd.edu.au> 09/06 10:33 PM >>>
Dear all,
I have a question about deciding which model describes one's data best. I
have recorded sedimentation equilibrium data at three speeds with three
different dilutions for a protein which undergoes some self-association. I
have been fitting the data in NONLIN. If i fit the data by fixing sigma to
the monomer mass, allowing delta y and lnA values to float, and permitting
a single association constant to float, i get the best fit with a
monomer-trimer model (both monomer-dimer and monomer-tetramer have worse
residuals and higher chi-squared etc according to NONLIN). If I instead
allow an extra equilibrium constant to float, and call the two
associations
monomer-dimer and monomer-tetramer, i get a slightly lower chi-squared
(0.0138 vs 0.014 for the monomer-trimer model). My question is, how do i
decide if the extra complexity of the model is justified. I know there is
a
thing called an F-test, and thought that might be appropriate. If so, how
does one apply it in this case? What do you all do in these situations? It
seems that the extra variable is pretty risky, but presumably a
sufficiantly large reduction in the chi-squared would justify its
inclusion.
cheers and thanks in advance for any help,
Joel Mackay
************************************************************************
Dr Joel Mackay			ph +61-2-9351-3906
ARC Research Fellow			fax +61-2-9351-4726
Department of Biochemistry
University of Sydney
NSW 2006 Australia
http://www.biochem.usyd.edu.au/~joel/ 
************************************************************************