Antibody Isolation Problems

Mark J. Koebbe mkoebbe at pond.com
Sat Oct 24 12:59:29 EST 1998


Dear Group,

Any help on this problem would be GREATLY appreciated.  We have serum (a
fair amount) from a rabbit immunized to a translated (HIS tagged)
peptide.  Nickel column purification yielded a fairly large amount of
protein (too large in my estimation).  The 'antibody' in that fraction
seems to be very 'dirty' in use with conventional immunohistochemistry
(I've used biotinylated secondaries and divalent metal
intensification).   I am unconvinced that we have an adequate amount of
antibody.  The possibility that the antibody (if it is present) has been
modified by purification has occurred to me.  I would like to recoup any
possible antibody with a minimal disturbance of the native conformation
of the antibody.

I'm proposing to take serum and do hydroxyapatite purification of the Ig
fraction as a first step.  Then continue with the nickel column.  The
hopes being that a pre-purified Ig fraction will have less potential
complications on subsequent purification.  I have also considered
Protein A as a method to isolate the Ig fraction.  As I have never
performed these two procedures (I have tried nickel column), I would
appreciate any advice.

Any help in the form of E-mail (my address is real) or posting to the
group would be highly highly appreicated.

Mark J. Koebbe




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