I have a set of buffers (Tris, imidazole, phosphate) for NI-NTA protein purification. I prepared them about three weeks ago and they contain DTT and PMSF. Can I add fresh DTT and PMSF to the buffers and use them or do I need to prepare a new set of buffers?
You can use them after adding fresh DTT and PMSF as long as they are not contaminated. You can also filter them with 0.2 filter before use. You can use Beta mercaptoethanol instead of DTT because DTT may not be compatible if DTT concentration is very high
I agree with Mahesh Chandak You can use them after adding fresh DTT and PMSF as long as they are not contaminated
Be careful that the total concentration of DTT does not exceed the maximum concentration recommended by the manufacturer, since excess DTT may reduce the Ni in your column. Personally, I would make the buffers fresh starting from the stock solutions of Tris, imidazole, and phosphate and add DTT + PMSF just before use
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