Hi,

after Protein-A dilute Mab with a 10-20 mM citrate buffer pH 5.0, them apply directely to S-sepharose ( from GE), wash with same buffer them elute Mab with NaCl 150 mM or high.

regards,

Fernando

for CaptoAdhere (combined IEX-IHC columns from GE) just download this from GE: http://wolfson.huji.ac.il/purification/PDF/IonExchange/AMERSHAM_CaptoAdhereInstruct.pdf

and followed the protocol for elution.

Fernando, thanks a lot for your help.

I have the column instructions,

but I'd like to receive some direct experience on it, like ‘I polished successfully a mouse IgG1 from leaked Protein A, using the buffer xxx’ or ‘I purified a rat IgG2a from endotoxins using these conditions xxx’,

- or ‘you can use these conditions for all the mouse IgG1’ or ‘ you need to setup the conditions for each molecule’

- or also ‘I tried to polish this IgG, but this support doesn’t work so good, butyl-S-sepharose (e.g.) is better (e.g.)’…

(probably I was not so clear in the post, I'll slightly modify it).

The matter is that –at the moment- this second step is still only an idea, I still don’t know exactly what I’ll need to eliminate (Protein A, dimers/aggregates, endotoxins, or other contaminants present in culture medium (e.g.) with recipes usually not published). So I’d like to collect some experience (exactly as you did in the first reply, but a. now, I don’t have the S-sepharose b. I’d prefer a ‘flow-through mode’ process), prior to start with the 8-11 experiments (DoE) needed to define the best conditions for my molecule.

thanks again!

You should made some trial tests with your sample, with a samll amounts, like 20 to 50 to ml of medium them when you got it optimized used a bigger sample like 500 to 1000 ml.

I used protein A and elluted the mouse igG according to the IgG isotype. I also made some extra modifications like wash with ph buffer that don't ellute the antibody but remove other proteins. For example if with protein A, the IgG1 elute for exemple at pH 5, I will do a previous wash with ph 7, and they recommend and them another wash with with pH 5.6,and them finally to ellute the antibody I will use the correct one to elute the IgG1 from Protein-A.

I never used the CaptoAdhere, only S-Sepharose or Abx from Bakerbond to polish.

look in this paper for ideias: http://144.206.159.178/FT/553/206368/5189512.pdf