Dear Vít Vykoukal ,

I suggest you to read the following (just search in Google Scholar and you will find a downloadable version):

Bartling, K. (2006). Apoferritin crystallization in relation to eye cataract (Doctoral dissertation, Georgia Institute of Technology).

Here you will see a wealth of practical tips and references. Like the de-ironization step at pH 4.5.

Hope this helps you a bit further.

Best regards.

Hi Vykoukal

Part of the reason why your metal ion of interest does not get caged by apoferritin molecules is simply their much lower dissociation constant at near-neutral/physiological pH. There are two ways to address such problems: 1). mild denaturation of protein with a non-chelating substance like 3M urea or guanidine hydrochloride, and 2). Gradually reducing the pH of the protein solution, incubation with the metal salt of choice, and then bringing the pH back to neutral to help in the association, all the while maintaining the temperature at ~4oC.

Required fraction, most probably at a molecular mass of ~475 kDa, can be isolated using size exclusion chromatography using an appropriate column like Superose 6 (GE). You will lose some protein, but you should still get enough for grid preparation.

Hope this helps.