But yeah, keep it cold during the prep, use omp-/lon- bugs, and if your purification protocol is good enough you shouldn’t have a problem. I always use excess PMSF when doing the bug lysis, but don’t add it after that. One ion exchange and one gel filtration is usually enough to purifiy away from everything, including the vast majority of proteases. Stuff that gets in after that is likely to be from bacterial contamination of the purified protein.

And I used to work on nuclear pore FG repeats which are notoriously susceptibe to proteolysis, so I think I know whereof I speak.

I never used them when I was purifying proteins routinely because the volumes I typically worked with were too large and I was doing drug studies with NMR and didn’t want to even think about how they may alter the interaction of drugs with my protein. Just keeping everything cold was sufficient . When I started doing ChIP assays they became more relevant because we would do many steps out of the cold room.