purifying antibody in small volume

[Pow Joshi]

On 4/30/07, fabianh from umich.edu <fabianh from umich.edu> wrote:
>
> Hi,
>
> Does anyone know of a way to purify antibodies resulting in small (and
> concentrated)volumes?
>
> I got an antibody which is not very strong. I have to use it in a 1:800
> dilution at the most to detect anything on my blots. the problem is
> that I also detect several other proteins. I tried to purify it by
> incubating with e.coli celllysate of a nullstrain for my protein and it
> worked quite well (only one band left) but now it is even weaker as
> this protocol uses SDS and a total volume of 10ml for 100ul of
> antibody. So my purified antibody is diluted 1:100 already leaving me
> with a 1:8 dilution (which then needed a 45min exposure of the film to
> detect weak bands)! And i would need a really strong signal for my
> purposes.
> Thanx a lot for any help




hi Fabian,

it seems to me that your antibody is of a low avidity/ affinity. A few tips:
when you do any kind of purification step, check the ab solution pH .... low
pHs will slowly degrade it, and your binding will not be as good

you could also try protein A/G/L purification step .... I have found that
this enriches the antibody fraction in the serum......you can probably use
small columns (say 3 ml) if you have say 1-2  ml of the post-e coli binding
step.

the decision of which of the A/G/L to use would depend on the host and
isotype of the antibody ..... I could send you a table for that.

and ofcourse, you can always have additional steps as Mike suggested, like
the ammonium sulphate pption, or even an ion exchange chromatography,
before the protein a/g/l step ..... however, these would be helpful only of
you have fairly large amounts of starting serum (I would say at least 3-5 ml
)

hope that helps,

pow

Fabian
>
> Bardwell Lab
> University of Michigan
>
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