Robert,
I haven't made MABs with PAGE eluted proteins but I have made polyclonal
antibodies with them and the antisera I generated had an excellent specific
titer. I used it at a dilution of > 1 : 5000 for Western blots. The antisera
however, could not be used for immunopercipitation. At the time we
hypothesized that the sera was reacting to the linearized protein and that
little if any secondary protein structure had reformed after elution from the
gel.
While doing this work we considered using the whole gel, but we didn't think we
could get the protocol passed the Animal Safety Committee so we didn't bother
trying. From what I remember, the thought was that the SDS and the acrylamide
would cause severe lesion at the point of injections and cause undue suffering
for the animal.
If you want our protocol let me know and I'll try to dig it up.
Bart Corsaro
Lederle-Praxis Biologicals
West Henrietta, New York
EMail: Bart_Corsaro_at_USLRMG01 at internetmail.pr.cyanamid.com
_______________________________________________________________________________
My question: Has anybody raised Mabs using protein bands excised
from PAGE gels as the immunogen? If so can you use the gel band
itself as an implant without having to elute the protein?
Second question ( I have asked this before but for the benefit of
newcomers) Is anybody interested in Fungal diagnostics by serological
methods?
Robert
Robert Burns
Monoclonal Antibody Unit
Scottish Agricultural Science Agency
East Craigs
Edinburgh
Scotland
burns at sasa.gov.uk
