densitometry on western blots

martinhoehne At gmx.net via methods%40net.bio.net (by martinhoehne At gmx.net)
Mon Nov 27 11:41:20 EST 2006


Thanks for the input. Regarding the equations cited below,

> With some caveats. Essentially, you're taking the simple equilibrium
> binding reaction:
>
> A + B <=> AB

our working hypothesis is indeed as simple as this.



> And its corresponding equation:
>
> Keq = [AB] / [A][B]
>
> ............
>
> In practice, the latter two conditions require that both proteins are
> present in substantial excess over the complex, which in turn requires
> that the affinity of the interaction is fairly low. For example, you
> could probably measure a kinase binding to a scaffold like this, but i
> think you'd be on thin ice measuring an antibody binding to an antigen.
>
> Disclaimer: i don't do this stuff for a living, and it's years since i
> studied it as a student, so i could have this wrong. Does this make sense
> to anyone else?

I don´t know if I got this right.
My naive thoughts were:
Since I´m precipitating protein A_wt and A_mut and want to know if one
co-precipitates more protein B than the other, I thought that it´s
only necessary (and maybe even better for this kind of exp.?) that
protein B is present in excess. Wouldn´t that ensure that protein A
can bind and coprecipitate as much protein B as possible (for the given
mutant).
To see whether protein B is really present in excess I thought I simply
can take an aliquot of the supernatant after pelleting the IP-beads and
check if there is still protein B in solution that is not precipitated.
Is this a valid method? 

Thanks in advance, Martin



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