I have to start the purification of a protein using a NI-NTA superflow column. My protein has an aproximate pI of 7. I have no experience with IMAC purification, i know i may have to try different conditions, but i would like to know which binding buffer would you recommend? What pH, salt concentration, presence of imidazole (10mM)? Thanks!
You mean the buffer that your protein will be on while passing on the column? You will have to choose according to your protein, a pH of 7,4 or 8 (like Tris or HEPES 50 mM), you can choose to add imidazole if your protein supports it and it still binds to the column (like 10-20 mM), NaCl (100-200 mM as a starting point, after that you may have to increase or not). Moreover, you can add compounds that may be needed for your protein (detergent if a membrane protein, TCEP, glyerol (depending on the downstream applications). If you are using other compounds, verify the compatibility with the Nickel column
So a good starting point would be a 50 mM HEPES or Tris pH 7,4 , 150 mM NaCl , 10 mM imidazole (?) .
Thank you very much for your answer! I will try with that buffer.
There is no general rule for anything in imac as it highly depends on your particular protein. Some general comments:
1. 150 mM NaCl is not enough to completely forbid electrostatic interactions, use at least 500 mM.
2. Use phosphate buffer at pH 7, it is way cheaper than tris or hepes.
3. 20 mM is more than enough for buffering unless you have an extraordinary high protein concentration
4. Imidazole contributes towards selectivity, nonetheless Co(II) or Zn(II) might give better results.
5. Even for recombinant 6xhis-tagged proteins, purification with imac is not straightforward.
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