Dr Engelbert Buxbaum <engelbert_buxbaum at web.de> wrote:
>"D.K." wrote:
>>> - Purify IgG on Protein A, elute with 100 mM Glycin, pH 2.5,
>> immediately neutralize to pH 8.0 with 2 M Tris, pH 9.0,
>> add NaCl to 0.35 M final.
>> - Run over an antigen column, load 2 times to ensure saturation
>> of binding sites, with 3.6 M MgCl2, pH 6.5. Dialyse extensively,
>> make sure to leave room for water in dialysis bag. MgCl2
>> elution is a lot milder on most native proteins than standard
>> acid elution.
>>Elution with caotropes from an affinity column selects for mediocre
>antibodies, as the realy high affrinity ones stick or elute at
>concentrations of denaturants, which are destructive.
Not in my experience. Various polyclonals that I purified
using the above method were extremely inhibitory in functional
assays (yes, controls with IgG from depleted serum were always
done). I routinely analyse what elutes from the column(s)
during regeneration pH 1.8 and there was never any more than
5% of what I eluted (I do let the column sit for 15 min in MgCl2
between elutions with 1 column volumes).
DK
