Hi Theresa: If you can make sure that your target protein is expressed. You can first use 6 M urea to denature the protein and then try to bind it to the column. If the denatured protein can bind to the column, it seems the histag is hided inside of the protein. It is not exposed enough to interact with the column. In this case, you can design a new construct, for example, put the tag to the other end of the sequence, or introduce a flexible linker between the tag and the protein. Another thing you can try is using Cu ion instead of Ni ion. It will fasten the binding. Good luck Yu Xiaodi
> Date: Sun, 15 Jan 2012 18:23:33 +0000 > From: [email protected] > Subject: [ccp4bb] Off topic: His-tag purification > To: [email protected] > > 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
