>
>I thought about HIC too, but do not know if it would work since the
>binding of protein to HIC need high salt conc. and I am not sure if the
>salt conc. in the sf900 or Hi5 medium is high enough (the formulation is
>secret, LOL), thus it is good to know that someone has succesful
>experience with HIC.
Few things:
1. With regard to the salt/ionic strength, the formulation of serum-free
media cannot be far off from the traditional media. Very crudely speaking,
figure an equivalent of ~200 mM NaCl. So most protein won't bind to, say,
phenyl sepharose under these conditions. But "your" protein might be in the
minority - who knows?
2. What prevents you from adding extra salt to the collected medium? Say,
~1 M ammonium sulphate final? There probably will be some precipitate
forming which can be filtered away before loading onto a HIC column.
3. Hydroxylapatite. "Ceramic Type I" version from Bio-Rad in particular.
Large size beads packed into a wide column. A great way to concentrate
total protein at high flow rates. Phosphate concentration in the medium is
low enough that majority of proteins will sill bind.
- Dima
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