protein affinity purification

Dima Klenchin klenchin at facstaff.wisc.edu
Tue Sep 24 09:34:53 EST 1996


In article <199609231357.JAA11376 at mail.med.cornell.edu>, mf at aol.com (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 stud.med.cornell.edu

pH 2.2 will work for sure (if your complex is OK with it). 
Alternatively, chaotrops (thiocyanate, etc) will work too, but the 
column will deteriorate even faster than with low pH. 
Reverse elution isn't a bad idea but you will have considerable "tailing".

Good luck,

- Dima



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