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. Institute of Toxicology Tel.: 089-3187-2669 Fax: 089-3187-3449 www.helmholtz-muenchen.de/en/toxi/krappmann<http://www.helmholtz-muenchen.de/en/toxi/krappmann> ________________________________ From: "Theresa H. Hsu" <[email protected]> To: [email protected] 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 ________________________________ Helmholtz Zentrum M?nchen Deutsches Forschungszentrum f?r Gesundheit und Umwelt (GmbH) Ingolst?dter Landstr. 1 85764 Neuherberg www.helmholtz-muenchen.de Aufsichtsratsvorsitzende: MinDir?in B?rbel Brumme-Bothe Gesch?ftsf?hrer: Prof. Dr. G?nther Wess und Dr. Nikolaus Blum Registergericht: Amtsgericht M?nchen HRB 6466 USt-IdNr: DE 129521671
