Question

3. The Ni-NTA protocol for this week differs from that of week 2 in that a different buffer component will be used to release your protein from the column. What chemical in the elution buffer will achieve this? Why are we not using imidazole for this step?

Answer 1

Any reducing agent, chelating agent [or too high imidazole] in your buffer binding of your protein will be reduced or even impossible. So this technique is very handy. Alternatively you may use following buffer to release your protein from the column. The composition of buffer is 50mM phosphate (pH-8.0), 300 mM NaCl, 1mM PMSF, or even if you increase NaCl concentration then it will release your protein from the column.

Free imidazole has been known to denature protein when freezing and thawing. Adding imidazole to your buffer, will change the pH of the solution. Imidazole has a higher affinity for the metal than does histidine. Therefore, it is important to continue to the next chromatography before freezing sample.

Hope this helped you!

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