I am having problem purifying his-tagged protein using Talon cobalt resin. I can purify same protein Ni-his but I get some none specific binding proteins in the elution fractions. Cells were lysed using bugbuster master mix and 1mM PMSF was added. I passed the supernatant in the column Talon resin and I have nothing in the elution. I used the same buffers as for the Ni-his purification.
Equilibration/Binding buffer: 20mM Tris/HCL, 200mM NaCl; pH 7.5
Wash buffer: 20mM Tris/HCL, 200mM NaCl, 5mM imidazole; pH 7.5
Elution buffer: 20mM Tris/HCL, 200mM NaCl, 250mM imidazole; pH 7.5
The protein has both N- and C-terminal histag.
Did you make sure you protein expresses at all (gel of non-induced vs induced culture, and pellet vs lysate after bugbuster)?
Yes, I have checked this, I can see the protein in the induced culture and lysate after bugbuster no protein in the pellet.
Do you know the structure of the protein? I wonder if the terminus where you put His tag might be occluded. I'd try switching His-tag to the other terminus if possible, alternatively using a longer linker. You use a very low concentration of imidazole in your wash, so the protein clearly just doesn't bind. It's definitely not bugbuster's fault, I've successfully used it for dozens of protein preps with Ni and Co NTA resins.
Thank you Katarzyna Radziwon ,
I have hist tag on both sides of the rec-protein, so I think if one side is inaccessible, the other side should atleast accessible.
What of the buffers (binding/equilibration, wash and elution) that I am using? could they contribute?
For one protein, for me to even Ni-hist purify, I had to add 8M urea to denature the protein as both hist-tags were buried in the native protein (After this was able to purify this protein).
You can try increasing pH to 8.0, that definitely improves binding, but 7.5 should be okay too. If possible, run it on His trap on AKTA with gradient.
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