Hi Theresa, I am right in thinking the protein construct you are using is just 8 His residues added to 90kDa ? It could be (if this is the case) that the tag is not accessible for binding to the Ni column. Sometimes you need a linker sequence between the protein for the His-tag to coordinate well to the Ni2+.
Hope that helps Cheers Jodie ________________________________________ From: CCP4 bulletin board [[email protected]] on behalf of Theresa H. Hsu [[email protected]] Sent: Monday, 16 January 2012 7:23 a.m. To: [email protected] Subject: [ccp4bb] Off topic: His-tag purification Hi all I have a His-tagged soluble protein (8 His residues added to 90 kDa protein) that do not bind to IMAC column based on flowthrough showing up with Western blott. Do you have suggestions to improve the binding? Binding condition is 50 mM Tris-HCl 8.0, 300 mM NaCl, 10 mM imidazole pH to 8.0. Thank you. Theresa
