In article <48qeib$i6l at highway.LeidenUniv.nl>,
ron at ruly46.medfac.leidenuniv.nl (Ron Smits) wrote:
>We have raised several rabbit polyclonal antibodies against fusion proteins of
>aminoacids. All of them recognize on westerns the expected protein but as
expected for polyclonals,
>also a lot of extra bands (specific aspecific).
>When we tried to get rid of all these extra bands with affinity columns, the
pictures on western were
>cleaner with respect to background but not for the aspecific bands. Does anyone
know why we
>can't get rid of these aspecific bands? Is there a protocol for purification which
has more chance of
>We have thought ourselves of using proteinextracts of tissue which doesn't
contain the protein of
>interest, to compete out the aspecific antibodies. Some people told us this does
work in principle but
>not in practice. Is there anyone who has some success stories on this matter?
>Any suggestion is welcome!
First, titrate your serum. In my experience, the min. dilution to start with is
1:1000 and up to 1:20000. Try to dilute and see if it helps. Worked for me.
If it does not, then you've got a problem.
Two suggestions. (Never tried both personally, but know people that used them
1. Usually, for small scale (like western blotting) the purification could be done
on blot: EF lots of your protein, blot it, excise your band, block and incubate the
strip with your serum; wash and elute Ab with low pH, neutralize. The band from
1-comb minigel could be cut in piecies and eluted in 0.5-1 ml. Dilution for Western
is in the range of 100-1000. The blot could be reused, I think.
This, in principle, is the same as you've done on a larger scale - affinity
purification, so it may not result in disappearance of the "nonspecific" bands.
2. Depletion that you've mentioned. Run your serum through a sepharose to which
a track load of your protein is linked (easy to do yourself, but, I think, Sigma sells
total E.coli protein-sepharose).
Hope it helps,
/ /\ Dima Klenchin
/ / \
/ / /\ \ klenchin at macc.wisc.edu
/ / /\ \ \ tel. (608)262-4380
/ /_/__\ \ \ FAX (608)262-4570
/________\ \ \