Hi, Shubhangi, One possibility is that the his-tag could be non-accessible. Have you tried moving to the tag to the other end of the protein (N vs C)? Another thing that worked for me for a past project is using a small amount (1-2M) of urea for the IMAC step and then dialyzing away the urea before proceeding onto the other purification steps. This increased protein binding to the IMAC column and after dialyzing away the urea, the protein still retained full activity (I was also able to crystallize and determine the structure of it).
Hope this helps. --Eddie Edward E. Pryor, Ph.D. Anatrace Field Applications Scientist Cell: 434.270.2511 Find Anatrace on LinkedIn<https://www.linkedin.com/company/anatrace> From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Shubhangi Agarwal <shubhangiagarwal2...@gmail.com> Reply-To: Shubhangi Agarwal <shubhangiagarwal2...@gmail.com> Date: Thursday, March 30, 2017 at 3:20 PM To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] binding protein to nickel column 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
