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mAb Purification Difficulties

Walter Woith walter at see.sig.for.address
Thu Feb 6 05:58:44 EST 1997

David D. Shock dsho at med.unc.edu wrote:

> I'm attempting to purify a mouse monoclonal IgG1 from tissue culture
> supernatent with little luck. I'm using proten A sepharose (HiTRAP column
> from Pharmacia) and my yields are quite low. My protocol is as follows:
> Add NaCl slowly to the supernatent to reach 3M (no precipitation occurs)
> Adjust the pH to 8.0 by diluting supe 1:1 with 1M Tris-HCl, pH 8.0.
> Pass supe over a 1 ml HiTRAP column equilibrated with 50 mM Tris-HCl, pH
> 8.0, 3M NaCl at a flow rate of 1 ml/min at room temperature.
> Wash column until A280 returns to baseline
> Elute mAb with 0.1 M citric acid-NaOH, pH 3.5 collect 1 ml fractions
> supplemented with 250 =B5l 1 M Tris-HCl, pH 9.0

Dear David,
What's your problem: inclompete binding of your mAb to the affinity
matrix or insufficient elution? Have you ever checked your column flow
through for residual mAb?

> I've also tried protein G sepharose with  similiar results, ie low yield!

You probably know that mouse IgG1 has a relatively low affinity to
protein A and that's why you tried protein G. I use GammaBind Plus
Sepharose (Pharmacia) for several mouse IgG1 monoclonals with excellent
results. The protocol I use is straight according to Pharmacia's
instructions: binding and washing with 10 mM sodium phosphate, 150 mM
NaCl, 10 mM EDTA, pH 7.0; eluting with 0.5 M acetic acid, pH 3.0
(ammonium hydroxide).

Hope that helps


Walter Woith
walter.woith at med3.med.uni-erlangen.de
Fax +49 (0)9131 85-3399

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