protein extraction

Rod Savidge savidge at unb.ca
Fri Dec 1 10:44:57 EST 1995

We're thinking about using a monoclonal antibody (IgG) in an
attempt to immunolocalize a water-soluble molecule (MW <200) that
presumably exists in the cytosol or vacuole of plant cells.  We think we
can get the McAb into the cells OK, then cross-link the McAb-hapten
complex using a fixative to another protein (or other structure), and
follow with a secondary anti-IgG `reporter' antibody.  We know that
we cannot fix the hapten prior to introducing the McAb because it would
make the epitope unrecognizable.  Washing away unbound secondary antibody
should not be a problem; however, I can't think how - prior to the
cross-linking step - we can wash away unbound primary McAb without
concomitantly losing the McAb-hapten complex.  If anybody has experience
with this I'd appreciate receiving advice.
   Rod Savidge, PhD, Professor      |         E-mail: savidge at unb.ca
   Faculty of Forestry and         \|/
      Environmental Management  \   |   /     Phone:  (506) 453-4919
   University of New Brunswick  _\/ | \/_
   Fredericton, NB CANADA          \|/        Fax:    (506) 453-3538
   E3B 6C2                          |

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