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 [[log in to unmask]] on behalf of Theresa H. Hsu [[log in to unmask]]
Sent: Monday, 16 January 2012 7:23 a.m.
To: [log in to unmask]
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
|