Re: [ccp4bb] hydrohyapatite column
Hydroxyapatite can be miraculous, but is often very capricious: among others, it is temperature sensitive and two different corners of a somewhat inhomogeneous cold room may provide different results. Daniel Le 29/11/2010 16:08, Sebastiano Pasqualato a écrit : Hi all, I read/heard that hydroxyapatite column can be used to purify proteins, getting separation results orthogonal to ion exchange and size exclusion chromatography. I was wondering if any of you would be kind enough to share her/his experience with me, and would suggest vendors and models for such columns. Thanks in advance, best, s
[ccp4bb] hydrohyapatite column
Hi all, I read/heard that hydroxyapatite column can be used to purify proteins, getting separation results orthogonal to ion exchange and size exclusion chromatography. I was wondering if any of you would be kind enough to share her/his experience with me, and would suggest vendors and models for such columns. Thanks in advance, best, s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 9437 5990
Re: [ccp4bb] hydrohyapatite column
I was wondering if any of you would be kind enough to share her/his experience with me, and would suggest vendors and models for such columns. I really like ceramic hydroxyapatite from Bio-Rad. The only type that behaves in the columns long-term, without the need for repacking. It also gives better resolution and/or allows faster faster flow rates. Their type I has higher capacity and generally more useful. The great thing about hydrohyxapatite is that essentially all proteins bind to it as long as there is no phosphate around even in high salt (like 500 mM NaCl). So it is a great way to concentrate proteins from diluted solutions or a nice chromatography step right after AEX. Some proteins (generally few, mostly alkaline) will elute at very high NaCl (1-2 M), others with require phosphate. For ceramic hydroxyapatite I find that the useful range is 0-100 mM phosphate at pH ~ 7.0. Capacity of the ceramic hydroxyapatite is lower than the normal mineral form, only around 25 mg/ml. Dima
Re: [ccp4bb] hydrohyapatite column
Hi Sebastiano, Just to add to Dima's response, I've used ceramic hydroxyapatite column and got great results separating 2 very large membrane proteins complexes that eluted together in DEAE/Q and ran in similar size range in SEC. One eluted in 100-200mM KPi and the the other in 300mM+ KPi ~pH 7.4. -john On Mon, Nov 29, 2010 at 9:08 AM, Sebastiano Pasqualato sebastiano.pasqual...@ifom-ieo-campus.it wrote: Hi all, I read/heard that hydroxyapatite column can be used to purify proteins, getting separation results orthogonal to ion exchange and size exclusion chromatography. I was wondering if any of you would be kind enough to share her/his experience with me, and would suggest vendors and models for such columns. Thanks in advance, best, s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 9437 5990
