You could try directly binding the antibody to protein A-coupled Sepharose
(Repligen) using dimethylpimelimidate (Pierce) as crosslinker and eluting
the antigen with a low pH buffer; we use 0.2 N acetic acid. If Edman
sequencing is involved, do not use glycine!! If the protocol for preparing
the affinity matrix is followed to the last word, it should work like a
charm. Very little antibody (heavy or light chain), if at all, elutes from
the column; one could hope for at least 80% pure antigen by this method; we
have purified proteins (MHC class I antigen presenting molecules) to close
to 95% purity. If the antibody binds poorly or not at all to protein A,
protein G (Pharmacia or Pierce) or protein L (Pierce or Sigma) can be used.
Repligen's recombinant protein A-coupled Sepharose has worked best in our
hands; they claim that the serum albumin-binding domain/region of protein A
was deleted while engineering the recombinant protein. Details of chemical
coupling of antibody to protein A or G Sepharose (I suspect that coupling
to protein L will also work by this method) can be found in:
Harlow and Lane's "Using Antibody: a laboratory manual" Cold Spring Harbour
Laboratory Press, Cold Spring Harbour; pp 321--325, 1999.
{THIS MANUAL IS RECOMMENDED TO ALL THOSE WHO USE ANTIBODIES IN THEIR
RESEARCH.}
If you do not have access to this manual, I could send you a facsimile copy
of the relavent pages.
Good luck!
With warm regards,
sebastian
Sebastian Joyce, Ph.D.
Department of Microbiology and Immunology
C6804C, H107
Pennsylvania State University College of Medicine
Milton S. Hershey Medical Center
500 University Drive
Hershey, PA 17033-0850
Telephone: 717-531-4163 (office)
717-531-4181 (laboratory)
Facsimile: 717-531-4600
Email: sjoyce@psu.edu
sxj14@email.psu.edu