[Protein-analysis] Re: affinity columns
(by allison from nospam.com)
Fri Mar 30 16:40:06 EST 2007
Dan Guire wrote:
> Hi Allison,
> I'm trying to prepare an immobilized-antibody affinity column. I've tried
> using a commercial kit that includes a gel which is intended to bind
> antibodies that have been pretreated with a reagent that's included in the
> kit. My problem is that in the course of troubleshooting the procedure, I
> found that just exposing the gel to the antibody solution without
> pretreating the antibody with their reagent is enough to confer affinity to
> the gel. It binds my antigen about as well as if I had used their reagent,
> so the antibody must be passively adsorbed there to a significant extent.
> Columns prepared in this way can stand up to acid elutions and salt washes
> about as well too. So my question is: Is it possible that it's not
> preferable to covalently immobilize the affinity agent? I doubt that, which
> leads me to think that my method is misleading me.
> In testing the kit I've bought I've been using an enzyme-labeled protein as
> ligand. Since a small amount of enzyme can give back a large signal, the
> total amount of protein is small. Maybe I haven't been loading enough
> ligand onto the columns to reflect the true amounts of immobilized antibody
> on the differently-prepared columns.
> Hey I might try doing a BCA on the gels themselves to see if there's more
> protein on one than the other, or using a larger loading of an unlabeled
> version of my ligand. Any other thoughts will be much appreciated,
I haven't made my own affinity columns but some questions do come to mind:
Does your gel alone bind the test protein or does an Ab with a different
specificity also give the same effect? Because I agree with you that
enzyme-labelled proteins can sometimes be much too sensitive. Perhaps
the gel has some inherent "stickiness" and will bind a bit of Ab and a
bit of ligand without any treatment at all.
Does the kit come with any info as to how much antibody can be coupled
per gm of gel? If so, how does this compare with how much you can bind
What does the company that makes the kit say when you contact them?
Presumably they have lots of experience with troubleshooting and it is
in their best interests to help solve your problem for you.
Do you want to let us know the name of kit/company?
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