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Hi Theresa, You could try lowering the pH down to 7.4 or 7. I have found that some proteins bind very poorly at pH8 and higher. Hope this helps. Cheers! ------------------------------------------------------------------------------- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 -------------------------------------------------------------------------------- ** Hard work often pays of in time, but Procrastination always pays off now ** ----- Original Message ----- 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