Hi Theresa,
did you try to change the position of the His-Tag?
Perhaps it is not exposed in your current construct and would work better at the N- or C-terminus.
Greetings
Moritz Metlitzky, Ph.D.
Tel.: 089-3187-2669
Fax: 089-3187-3449
From:
"Theresa H. Hsu" <[log in to unmask]>
To: [log in to unmask]
Sent: Sunday, January 15, 2012 1:23:33 PM
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