Antibody Problems

Dear All

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?

Thank you in advance.

In article <[Only registered users see links. ]>, patingsadagat <[Only registered users see links. ]> wrote:

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.

DK

On Tue, 8 Jan 2008, DK wrote:


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) [Only registered users see links. ]

In article <[Only registered users see links. ].ac.uk>, Tom Anderson <[Only registered users see links. ].uk> wrote:


That's why no one should ever decide on immunizing a rabbit
without pre-screening.


That would be almost criminal. Always demand "final bleed"
serum (which should be, in volume, on the order of 5% of body
weight).

DK

The protein I'm studying does not have any homology to tubulin.

I performed an experiment exploiting the fact that my recombinant
protein is 6his-tagged using Reacti-Gel from Pierce. However, I was
not able to get enough antibodies. I guess I can try this experiment
one more time using tubulin-blocked serum solutions.


I'm not sure if I can get them to do it all over again. It takes too
much time and I am trying to beat a deadline.

Thank you so much for your helpful comments and suggestions.

> I'm not sure if I can get them to do it all over again. It takes too

Hi,

In that case, as a fast and almost certainly working approach, you
might try to screen a phage display library displaying single-chain
antibodies (eg from NEB or the Tomlinson Library or whatever you can
get hold of) against your antigen. Then use the phages or the derived
single-chains (which usually have a His tag or so) to hybridize to
your blot. Detect with anti-His or anti-phage antibodies. Should be
practicable in less than 4 weeks.

Concerning the company which provided you that nice serum: They should
return you at least your money or even offer you or your lab three
free immunizations. With a prior check of the rabbits, as DK
suggested. Actually, that's so obvious. Why is nobody ever considering
that? One needs just a few drops of serum for this easy test.

Best of luck,

Wolfgang