I am currently having difficulty optimizing a binding buffer for Ni-NTA protein purification under denaturing conditions. My starting buffer is currently composed of 100 mM NaH2PO4, 1 M NaCl, 10 mM Tris-HCl pH 8, 8 M urea, and 5 mM imidazole; however, I am having large loss of protein immediately after incubation with the resin (initial run-through of cleared lysate through the gravity column followed by washes with binding buffer). Can someone recommend a fast initial screening protocol, where I do not need to complete the entire protocol in order to test a new buffer? The protein I am currently working on is a soluble 27.2 kDA lipoprotein for reference. Thank you!
You can work on a small scale by doing bulk binding. Mix the extract with a few hundred microliters settled volume of Ni-NTA resin in a microcentrifuge tube. Use a Nutator or other type of mixer to keep the beads suspended during the binding phase. Then spin down the resin. Transfer the supernatant. Add imidazole to the resin (same volume as the extract) and mix again. Spin down the resin and transfer the supernatant. Run all the supernatants on a gel and compare the 1st and 2nd supernatants for each condition to see how much of the protein was bound.
Dear Kevin An, Firstly you have to optimize the conc. of NaCl for binding buffer with and without imidazole. Because some time addition of imidazole restricts the binding of desired protein which is also contribute to loss of protein. Try to use Tris buffer not NaH2PO4. Optimize the conc. of imidazole in washing buffer from 10-50mM and also in elution buffer 100-350 mM. As you increase the conc. of imidazole, the purification will increase but concentration of protein will decrease so I suggest you t adjust the conc. of imidazole.
Are you working within the capacity of the resin?
The capacity of the resin is idealised and for any given protein can have a differing binding capacity.
I'm actually an advocate of using insufficient IMAC resin, as your protein of interest will compete off the non-specific proteins for a cleaner prep.
A SDS-PAGE gel would greatly help us understand your results.
If you are getting a nice fat clean band off your resin, then it's just a case of scaling appropriately.
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