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

Hi,

As mentioned above, DTT and PMSF can be added to the buffer without contamination, but the concentration of DTT and PMSF in the buffer is uncertain, which may result in the unrepeatable results of subsequent experiments, especially for the purification of proteins for crystallization that requires great attention to detail. My suggestion is to first configure the reservoir without DTT and PMSF, and then configure the buffer before the experiment and add DTT and PMSF proportionally.

Hope it helps!