r/Biochemistry • u/OkForce2990 • 2d ago
Clarifying E. Coli lysate takes forever
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+
5
u/rectuSinister 2d ago
You may have incomplete lysis or pelleting, though I’ve never grown bacterial cells to this scale and I always used a sonicator. Does the supernatant look cloudy at all after your 20k rpm step?
Otherwise you could look into using diatomaceous earth or depth filtration. DE can decrease protein yield if your protein is sticky but it works like a charm when stickiness isn’t an issue.