that was probably me that recommended the DynaBeads (that's the
Tosyl-activated Dynbeads) for immunoaffinity purifications.
We follow the manufacturers instructions for the couplings.
In contrast to your approach however, we ONLY couple affinity purified
antibody to these beads. I don't know what the molar ratios of specific
Ig's to total protein are but I suspect there are many orders of magnitude
difference between the concentrations of the albumins or carriers in your
preparations compared to the concentrations of the Ig's of interest. I
guess you've probably made some quite nice Dynabead-gelatin and
Dynabead-BSA preparations?
We deliberately raise anti-peptide antibodies (polyclonals in rabbits) so
we can immunoaffinity purify them against antigen prior to use. This
achieves several things, it concentrates and purifies the specific Ig and,
importantly, it selects out the Ig's with the sort of fast on-rate that you
need to achieve high affinity immunoaffinity chromatography. We have never
(in our limited experience) had success with monoclonals for this purpose,
we have never achieved the high affinities we can achieve in rabbits.
Our antibody purification is on peptide coupled to Pierce Sulpholink resin
in small open columns at 4 degrees with elution in pH2 glycine with
immediate neutralization, if you need a detailed protocol, I can send one.
When we raise anti-phosphopeptide antibodies, we always preclear the serum
by passing it over a dephosphopeptide column prior to purification.
Good luck....Ken
> Sometime in the last year, someone on our friendly network
>mentioned that they were using dynabeads for immunoaffinity chromatography.
>We have been trying them for a couple of months now and haven't had any
>real success. I wonder if anyone out there has any suggestions or if the
>original person would share their protocol. We want to develop a general
>protocol for taking a commercial antibody aliquot and purifying enough
>protein to look for post-translational modifications by mass spec. Below I
>summarize what we have tried. Thanks in advance for any advice that the
>protein gurus out there might give us.
>
>
>We are using the protocol that the manufacturer suggests for the covalent
>binding of the antibody to the beads, with a polyclonal rabbit antibody to
>MAPK kinase from Santa Cruz (+ gelatin) and we have also tried a monoclonal
>from Transduction (+ BSA), both have yielded less than favorable results.
>Both antibodies have carrier proteins in them that we believe are
>interferring with the binding of antibody to the beads. But even increasing
>the amount of beads did not produce an improvement. A major problem is
>that there is no depletion of antibody when comparing the load and
>breakthrough of antibody. Also, when eluting all proteins off the bead we
>do not see any antibody heavy chain coming off with the carrier proteins.
>We have also tested the blocking and found that
>it is not sufficient. We also get very poor binding of purified MAPK kinase
>to the antibody-dynabeads, and what is binding is not eluting very well
>(probably because the blocking isn't adequate).
>
>We have used a variety of buffers to wash the beads and for antibody
>binding: Phosphate buffer pH 7.4
> Acetate buffer pH 4.0
> Borate buffer pH 9.0
>
>We have also used two different bead volumes
>
> first with 20ul of dynabead solution
> and later with 100ul
>
>We have varied coating solution volumes from 50ul-300ul, and decided larger
>volumes work better ie) 200-300ul
>
>We have blocked the beads with BSA and more recently with ethanolamine.
>
>We have also varied the amount of antibody added to the coating solution.
>Dynal recommends 1-3ug/1X10 7 beads. We have tried several amounts within
>this ratio as well as above and below.
>
>
>Katheryn Resing
>Dept Chem and Biochem
>Univ Colorado
>Boulder CO 80303
>
>303-492-6273
>fax 492-2439
********************************
Ken I. Mitchelhill
The John Holt Protein Structure Laboratory
St. Vincent's Institute of Medical Research
41 Victoria Parade
Fitzroy 3065 Victoria
AUSTRALIA
Telephone: 61-3-9288 2480
Facsimile: 61-3-9416 2676
Email: k.mitchelhill@medicine.unimelb.edu.au
Laboratory: http://www.medstv.unimelb.edu.au/WWWDOCS/SVIMRdocs/JHPSL.html
ABRF: http://www.abrf.org
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