M K Archer (fr80 at dial.pipex.com) wrote:
: We are trying to develop a western blot protocol without using
: SDS-PAGE and electroblotting. The method involves sucking protein
: through a nitrocellulose membrane in a 96-well dot-blot machine (usually
: used for DNA/RNA dot-blots). The antibodies are also added into the
: wells and sucked through the membrane.
So far, so good.
: Our problem has been we are unable to block the membrane
: sufficiently. The secondary antibody binds to the membrane even if the
: primary antibody is not present. We have tried a variety of blocking
: agents (BSA, Superblock-Pierce, etc.) but without success. Has anybody
: tried this sort of experiment or does anybody have any ideas.
Whoops! I think the problem is sucking the primary and secondary antibodies
through the apparatus. Antibodies are proteins, right? What's to
prevent them from adhering to the membrane just like the antigen?
After binding the antigen (sample) to the membrane, remove it from the
dot blot apparatus, dry it if you wish, then treat it in a floating bath,
roller bottle or Seal-A-Meal bag, just like a 'real' Western blot.
That should do the trick.
Assoc Prof Anatomy, Asst Prof Neurology (Research), Univ Mississippi Med Ctr
http://fiona.umsmed.edu/~hutchins/ *** E-mail: hutchins at umsmed.edu
``It became necessary to destroy the town in order to save it.''--American
infantry officer firing on Ben Tre, Vietnam, 2/8/68