protein affinity purification

Eric Roush edroush at
Mon Sep 23 13:39:02 EST 1996

In article <199609231357.JAA11376 at>, mf at (W) wrote:

> hey netters:
>    I've been working on a method to isolate proteins comprising a putative
> complex using an antibody to a single known protein in the complex. I've
> prepared polyclonal antibodies to the target protein, then I've affinity
> purified these antibodies and coupled them to a protein A bed (using DMP
> as a covalent coupling reagent). Then I've taken extracts from appropriate
> cells that we have previously shown express the target protein and passed
> them across the bed to isolate the "complex". At this point things have
> gotten a little difficult. Eluates from the column have shown by western
> that there is a very low yield of the target protein; moreover, when I've
> taken a small amount of the bed matrix and boiled in SDS-PAGE sample
> buffer, I get the target protein in expected amounts. Taken together I've
> interpreted this data as an indication that the elution conditions I'm
> using (0.2N Acetic acid, pH around 3.5) are not sufficient to disrupt the
> binding for long enough to get the protein off the column. I might add
> that at the point that I've affinity purified the antibody I used silver
> stained gels and ELISA assays to estimate the binding affinity (Kd) at 0.2
> nM, so it may make some sense that I'm disrupting and then rebinding as my
> protein moves down the column. I'm currently considering a reverse
> elution, as the idea here is that for a protein that binds with high
> affinity to the top lamina in a bed, the most effective elutions are
> accomplished by turning the column over and eluting in reverse. What I'm
> really looking for here is any advice that has worked in similar
> situations, along with whatever snippets of rationale that you may care to
> include. Thanks. jnbradsh at

How much buffer are you using to elute your column relative to
your column size?

How quickly are you running your column?

Have you been able to estimate a
k-off for your antigen-antibody complex?

Longer elution time and/or extensive use of elution buffer could
potentially improve your elution profile.

Eric Roush               Phil Niekro for the HOF!
edroush at   Any sufficiently advanced magic
also coache at      is indistuingishable from technology.
Maddux/Glavine in 1996!       It's Time for a Change-Up!

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