PS: if you are working with a human protein: most human proteins are quite stable at 37°C (for obvious reasons).
-----Ursprüngliche Nachricht----- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Schreuder, Herman R&D/DE Gesendet: Freitag, 27. Juni 2014 09:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] AW: [ccp4bb] How to remove phospholipids bound to a cytoplasmic protein Dear Lionel, In case of a lipid-binding protein, we were able to remove most (>90%) of the bound lipids with a lipidex column. This could be verified by DSF since the melting temperature of lipid-bound protein was ~6°C higher as the melting temperature of the free protein. The delipidated protein did crystallize but in the end we just replaced the bound lipid with our own compounds by adding these to the protein in the absence of the lipid. The first thing I would try in your case would be to set up crystallizations in the presence of a high (~10 mM) concentration of a natural ligand. Best, Herman -----Ursprüngliche Nachricht----- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Lionel Gesendet: Donnerstag, 26. Juni 2014 18:16 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] How to remove phospholipids bound to a cytoplasmic protein Dear all, I would like to remove two phospholipids bound to (actually into) a cytoplasmic protein. The protein was expressed in E. coli and the crystal structure revealed the presence of two co-purified phospholipids (most probably DPPE). A web search gave me three methods to remove bound lipids: - 1-butanol liquid–liquid extraction - Lipidex 1000, VI column at 37°C - HIC (aka Hydrophobic Interaction Chromatography) on a Phenyl HP column in presence of 1M ammonium sulphate All are described in Velkov 2008 (http://www.sciencedirect.com/science/article/pii/S1570023208002390). I assume these delipidation methods would also work for phospholipid; and I am more tempting by the last one, the milder one. I would appreciate any comment or practical advice. Best regards, Lionel
