Antibody Problems

Tom Anderson via methods%40net.bio.net (by ucgatan from ucl.ac.uk)
Tue Jan 8 12:55:25 EST 2008


On Tue, 8 Jan 2008, DK wrote:

> In article <467348b1-ee72-436f-a36a-5f32fa6d58da from v4g2000hsf.googlegroups.com>, patingsadagat <arnigambe from gmail.com> wrote:
>
> >I've been trying to produce antibodies against my protein of interest.
> >At first, I produced a His-6x tagged protein in E. coli and purified
> >the resulting protein using several purification methods. We then sent
> >the protein samples to a local antibody production company. After 2
> >months, they sent us sera from rabbits before immunization (0 W), 4, 6,
> >7 and 8 weeks after immunization. They also sent a protein A/G-purified
> >fraction of the 8W serum. However, when I used the serum for
> >immunostaining, I obtained unexpected localization patterns which was
> >similar to that of alpha tubulin localization in HeLa cells. I
> >therefore tested all fractions using tubulin protein and to my dismay,
> >all, including the 0W sera could detect the tubulin protein. I do not
> >totally comprehend just what is going on. Should I continue my attempts
> >to purify my antibody of interest using the recombinant protein (of
> >interest), or is this a futile exercise?
>
> Of course. Right now you are dealing with the total IgG fraction while
> the only reasonably pure antibody is the one purified on antigen
> affinity column. On a small scale, you can purify against your blotted
> protein band.

Right. I was about to ask if your protein had any homology to tubulin, and
could be inducing a cross-reacting antibody response, but the key thing is
that the pre-immune serum recognises tubulin, so it seems to be that this
particular rabbit has a tubulin allergy (well, except that'd be IgE, but
you know what i mean). I would imagine that affinity-purifying the
antibody would get rid of the cross-reaction; if you can try that with the
samples you have and repeat your IF/blot, that should assuage your
concerns. Doing it with the pre-immune serum, as a negative control, might
also be wise.

I think the fact that you have 6his-tagged antigen makes affinity
purification a lot easier, if you're prepared to expend some resin on it:
bind antigen to column, bind antibody to antigen, wash like the clappers,
elute the antibody from the antigen with thiocyanate, cross fingers and
hope that antigen doesn't come off too.

However, the existence of the tubulin reactivity is going to make life a
bit of a pain, since it means your purification has to be really, really
good. Or you'll have to add recombinant tubulin to your blocking buffer or
something. Any chance you can complain to the antibody company and get
them to do it again with a rabbit that isn't already making tubulin
antibodies? Maybe they're reusing rabbits! Also, if it's making a lot of
anti-tubulin antibodies, that might (might!) mean it's making less
anti-your-protein antibodies.

tom

-- 
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264   (f) +44 (20) 76797805   (e) thomas.anderson from ucl.ac.uk


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