Protein purification using mouse anti-Myc Tag IgG

Pow Joshi via methods%40net.bio.net (by pow.joshi At gmail.com)
Sat Dec 23 13:51:16 EST 2006


Hi Eberhard,

I found the table from Sigma ...and yes, it does say that for IgG1 the
binding is better with Protein G and L; and I presume you have the
IgG1 isotype.
I will give you the procedure I had used, a long time ago for such
purifications: However, for more details you could refer to Current
protocols in immunology and/ or Antibodies by Ed Harlow and David
Lane.

The way I went about my purifications was:
1. 45% ammonium sulphate cut off (use the precipitate for further
purifications, since that is where the bulk of the antibodies are)
2. I had added a step of DEAE sepharose step (I was using serum)
...however, I suppose you can delete this step.
3. I had made a small column (of about 1cm diameter and 4 cm height,
which would roughly make about 4 ml of bed volume) and equiliberated
the column with 10mM phosphate buffer, pH 7; applied the antibody
solution from step 2 on teh column; alllowed the antibody fraction to
bind for about 1.2 hr. Eluted the antibody with 100mM glycine-HCl
buffer, pH 2.7 (you will have to neutralise the eluate, otherwise the
antibody will be inactivated!)
You can concentrate this fraction further with ultrafiltration.
You will have >=95%  purity.

I would imagine, that for you, even the ammounium sulphate
precipitation should work well, since you have serum free culture
supernate, and the only protein there would be the monoclonal released
(perhaps a few cytokines maybe?)
let me know if you'd have any further questions.....and hope that works for you.

pow


On 12/23/06, Breslauer <breslauer At wp.pl> wrote:
> Hi Pow,
>
> Thanks for reply. According to my exp: I will (or better: I hope) to bind
> mice anty-Myc  antibodies from hybrydoma serum free culture supernatant to
> kind of resign (there are at least two commercial resigns of my inrerest:
> Protein G- Agarose from Qiagen and magnetic Sepharose or agarose with
> Protein G from Invitrogen). Of course both producers claims that binding
> capacity is very high (up to 2.5mg/ml of resign for human IgG). I found
> these tables you mentioned it says first  of all that my mice IgG is
> subclass 1 thus I should use ProteinG. But the main problem I have to face
> up is if it is reasonable to construct such a column eg agarose - Protein G-
> anti-Myc to effectively pufify my mycTagged protein. I'm little confused as
> haven't find any publication yet in which such an  approach would be
> investigated. Have _you_ heard about it?
>
> saying hello from Poland, Eberhard
>
>
>
> Sure.... I would use Protein G for rat antibodies.... the binding is better
> for those (I had used ProtG for rat ones, successfully)
> sometimes, depending on the isotype, you could use a combination of prot A
> and G together.
> You could do a test run and check out how the recovery is..... let me know
> if you have specific questions.
> There is a table somewhere telling you the binding capacities of each,
> Protein A/G/L .... I shall try to find it and post it on the biomednet site.
>
> best,
> pow
>
>
> On 12/22/06, Eberhard Mock <breslauer1981 At gmail.com> wrote:
>  Hi there,May we discuss via mail my post u replied yesterday (it was about
> my idea puryfing protein using resign with Protein G and anti myc
> antibodies.
> Looking someone who has experience in such a stuff.
> Bres
>
>
>
>
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