[RASMB] Help needed

[Borries Demeler]

On Thu, Aug 23, 2012 at 04:21:49PM -0400, Ewa Folta-Stogniew wrote:

> _______________________________________________
> At 03:44 PM 8/23/2012, Sophia Kenrick wrote:
>
>   Depending on the kinetics of the reaction, you may be able to measure a
>   higher molar mass at the very apex of the peak (where the concentration is
>   highest) as compared to the leading or trailing edge where the
>   concentration is lower.  However, often you get a constant, average Mw
>   across the peak that changes as a function of the amount of protein that
>   was loaded on the column.
>
>   and:
>
>   You can get a semi-quantitative estimate of the K[D] from this type of
>   experiment.
>
>   the last sentence is not true; depending on the kinetics of the
>   association, you can get quantitative estimate of Kd for self-association-
>   you just need to prove that this is the case.
>
>   Wouldn't the kinetics of self-association be a concern for use of  sed.
>   velocity for Kd determination as well?
>
>   Ewa

It's not really a concern when all you want is a Kd. For purposes of
determining rate constants, the question is whether the reaction is too
fast compared to the time scale of the velocity experiment. Whether
it is or not depends on the size of the molecule and the speed with
which you are running.  If the reaction is too fast, you simply cannot
determine the rate constant of the reaction, but the Kd will still be
measurable. In the extreme case of non-interaction, you will simply
get two boundaries corresponding to monomer and oligomer.  (for kd
calculation you need molar concentrations, and you need to keep in mind
that the dimer absorbs twice as much as the monomer)

In the other extreme, when the reaction is faster than the time scale
of the vcelocity experiment, you get a reaction boundary, which changes
gradually from monomer towards oligomer as you go up in the concentration
gradient. In my experience for a typical protein the upper limit of a
k_off you can measure in a velocity experiment at 60krpm is a rather
slow speed of about 10^-4/sec. For all reactions, the Kd should
still be easily measurable as long as you have enough signal from
both the monomer and the oligomer.

-b.