I have a similar issue because I have a lot of protein in the unbound fraction. Maybe the his-tag is not well exposed... Have you tried to use Ni-NTA beads and binding overnight? How long is your his-tag?
Best, Chiara ________________________________ Da: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> per conto di Mark J van Raaij <mjvanra...@cnb.csic.es> Inviato: sabato 1 aprile 2017 11.46 A: CCP4BB@JISCMAIL.AC.UK Oggetto: Re: [ccp4bb] binding protein to nickel column Could it be due to the fact that the protein is a histidine kinase - perhaps it is interacting with or even modifying its own his-tag? Mark J van Raaij Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC calle Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://wwwuser.cnb.csic.es/~mjvanraaij Structural biology of viral fibres, CNB-CSIC<http://wwwuser.cnb.csic.es/~mjvanraaij> wwwuser.cnb.csic.es members (left-to-right) Mateo Seoane Blanco - doctoral student (FPI fellowship) Thanh H. Nguyen - doctoral student On 30 Mar 2017, at 21:20, Shubhangi Agarwal <shubhangiagarwal2...@gmail.com<mailto:shubhangiagarwal2...@gmail.com>> wrote: Hello I have a 10kda histidine kinase domain protein with a pI of 9.5. It has a C-term his tag and despite using different buffers the protein doesnt bind to the nickel cloumn. it comes out in the flow trhough. Buffers used- 50mM tris Ph=8, 300mM NaCl 50mM tris Ph=7, 300mM NaCl 50mM hepes Ph=7.5, 300mM NaCl 50mM tris Ph=8, 300mM NaCl, 10% glycerol Can someone suggest to get ensure binding of the protein to the nickel-nta column Shubhangi PhD student under Dr. Jhimli Dasgupta St. Xavier's College Kolkata India
