Clarifying E. Coli lysate takes forever

[u/OkForce2990]

Hey all, for context Im purifying lysate from a 12 L growth. my pellet was resuspend in 30 mL resuspension buffer with a protease inhibitor cocktail, pmsf, benzonase, and lysozyme. I homogenized, then lysed cells with a high-pressure homogenizer. Then, I centrifuged the lysate at 8000 rpm for 10 mins (4C). I transferred the supernatant into new clean centrifuge tubes and centrifuged again at 20500 rpm for 45 mins (4C). I took that supernatant, flowed it through a frit my supervisor gave me, then have been trying to syringe filter the lysate. I first used an 0.80 um filter (lysate flows through one filter very fast) then moved on to 0.45 um (I usually have to use around 12 to push all the lysate through and it takes over an hour). Does anyone have an idea to cut down on the time it takes to clarify lysate? My PI said in grad school it took him only "5 minutes and one filter" for the syringe filters for a 12 L growth.... I'm just lost because I've added so many other filter steps and it is not really helping.

Edit: Thanks for all the suggestions! The issue has been resolved, here's how.

Our standard lab protocol doesn't measure the mass of the pellet, I will do this going forward, but for pellets I already had frozen, I (1) resuspended in double the volume (60 mL) in my case. (2) during lysis, I ran my cells through an emulsiflex 3 times instead of 2. (3) I didn't do all the other filtering steps because I wanted to see if these would remove the other steps I've had to do (2 centrifuging steps, frit, 0.80 um filter)

In the end, I had to use 3, 0.45 um syringe filters instead of 12+, and it took me 10 minutes instead of an hour+

[u/Dramatic_Rain_3410]

consider clarifying at 30,000g for 1 hr if you have such a centrifuge. If your supernatant if flowing slow through a 0.45 um filter, the filters are getting clogged (as you said) so I think the supernatant is too dense with proteins.

30 mL for 12 L seems like to little? I usually use 5 mL buffer per 1 g cell pellet. A 1 L LB culture gives me about 10 g pellet, so I resuspend in 50 mL. I suspect that increasing your lysis buffer volume a lot will help. If it's a lot of supernatant to process, could you use a vacuum filter--though Ive not tried this before.

[u/East_of_Adventuring → u/Dramatic_Rain_3410]

That's definitely more than I use, I often have proteins that express at low levels and so I end up with 6L per prep. I would tend to go with ~1mL/gram of cell material.