densitometry on western blots

[Pow Joshi]

On 27 Nov 2006 08:41:20 -0800, martinhoehne At gmx.net
<martinhoehne At gmx.net> wrote:
> 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


if you wish to determine the near exact values, I suppose you will
have to determine them Scatchard analysis(say if it's an Ab-Ag
reaction), or Lineweaver Burke plots (or emzyme kinetics, or  by
non-linear regression analysis (perhaps if you want super accurate
results as this link claims:
http://www.graphpad.com/curvefit/index.htm) etc.
Frankly, I have never estimateed K(d) values on rountine basis,
however, you may find some curve fitting/data analysis tips in books
like Biophysical Methods by Cantor and Schimmel
(been almost a decade since I read those in school...so ca'nt tell you
where they are with respect to editions...but they are very useful for
things like Scatchard plots etc.) or regular Biochemistry text
books(Zubey, Lehninger are some of those).

hope this helps for starters....
pow

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