Antibody purification

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M.vanG
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Antibody purification

After antibody purification (using Protein G) we obtain aggregate formation during dialysis against PBS pH 7.4. We use Glycine-HCL pH 2.6 to remove the antibodies from the column. Followed by a dialysis step against PBS pH 7.4. Sometimes we obtain aggregate formation.
Has someone a tip to prevent for aggregate formation?

Add colour 2 ur life
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Hi,

Hi,
Here is a solution for your problem.
Elute the protein with Glycine Hcl buffer pH 3.5 and as soon as the protein eluted from the column neutralize the solution pH with 1.5M Tris pH 8.0 ~200uL, so that the final pH will be ~7.5 (titrate glycine 3.5 vs tris 8  and note down the ratio before you proceed to elute).

samm
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Hi Balu! You beat me to it!

Hi Balu! You beat me to it!
Remember to dialyse AFTER you follow the steps Balu outlined above. In fact, if you are using one of those little gravity columns, place the Tris pH 8 in your collection tube (s) - and voila - no more precipitates.

M.vanG
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We followed the advise given

We followed the advise given by Balu and add 1.5 M Tris (pH 8.2) to the antibody solution. pH before dialysis was 7.4. However unfortunately we still obtain a precipitation The purified antibody is a rat IgG2b. Has someone an other suggestion?