raising ANTIBODIES with fusion proteins??
spang at vms.biochem.mpg.de
Tue Sep 6 14:26:31 EST 1994
I do not know with which organism you are working, but I know you may get
a lot of unspecific bands wtith yeast proteins because of yeast
There are at leat 2 possibilities to purify the antibodies.
- Run your tagged protein on a preparative SDS-PAGE and blot it onto
nitrcellulose. Cut out the band and block it. Then incubate for 2h at RT
with sera, remove the serum and wash carefully with PBS. Elute the
specifically bound antibodies for 30 min with 100 mM Glycine pH 2.7.
Adjust the pH immediatly!!!!! Sometimes a Guadinium- hydrchloride wash
elutes further strongly bound antibodies.
-You may couple your tagged protein on Nickel agarose and immobilize the
protein with a covalent cross-linker like MBS. This column will be
resistant to a low pH elution with Glycine.
Hope it helps!
In article <fodde.13.0011D278 at ruly46>, fodde at ruly46 (Riccardo Fodde) wrote:
> We have made several his-tagged fusionproteins which we used to immunize
> rabbits. The sera (and also affinity-purified antibody) of these rabbits
> recognize on western-blots besides the expected band, also extra bands. Does
> anyone know a method to get rid of these antibodies recognizing the wrong
> bands? We are thinking of making subfragments of the original fusionprotein
> and then affinity purify the sera, hoping the aspecific antibodies don't
> recognize the subfragment but there must be more clever ways to do this?
> Besides this request for help I wonder what the current explanation is
> generation of these aspecific antibodies. All reactions and suggestions are
> Ron Smits
> Leiden University
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