[Protein-analysis] RE: Re: affinity columns (Allison)

Dan Guire via proteins%40net.bio.net (by dguire from isurtec.com)
Thu Mar 29 13:34:31 EST 2007


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,

DanG



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Today's Topics:

   1. Re: affinity columns (Allison)
   2. Re: Enthalpy of protein (Raoul Fleckman)
   3. Re: Enthalpy of protein (Frank K?ster)


----------------------------------------------------------------------

Message: 1
Date: Wed, 28 Mar 2007 15:27:46 -0500
From: Allison <allison from nospam.com>
Subject: [Protein-analysis] Re: affinity columns
To: proteins from net.bio.net
Message-ID: <qeadnT45_8AqXJfbnZ2dnUVZ_sjinZ2d from mcgill.ca>
Content-Type: text/plain; charset=us-ascii; format=flowed

Dan Guire wrote:

> Hi Barbara,
> 
> The main point has already been made:  Your anti rabbit alphatase
conjugate
> can only detect the rabbit antibody down on the Maxisorp plate.  It
doesn't
> matter whether there is antigen for the capture antibody or not.  No doubt
a
> negative control, leaving out only the mouse lysate, would show you that. 
> 
> The series Allison outlined looks fine; the enzyme-conjugated anti mouse
> shouldn't bind unless there is mouse antibody bound to antigen there.  My
> point is that if you already have biotinylated anti ENAC of one species,
you
> can capture any antigen with the non-biotinylated anti ENAC of the other
> species, then detect with the biotin and a streptavidin-enzyme conjugate.
> And a negative control is a must :-)
> 
> DanG
> 
> P.S. Do you have any affinity column experience by any chance?  I could
use
> some help in that arena...
> 
>

I have done a bit....what is your question?  If I can't help I am sure 
someone else can.
Allison


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