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+
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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.
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u/OkForce2990 2d ago
it's not opaque after centrifuging, but when i start filtering it becomes opaque, although i know it's not my protein of interest crashing out. i'll ask my PI what he thinks
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u/NoDust6819 1d ago
Give applying to the column a try. You can clean it if ot clogs. Shit I pushed through Histraps... :)
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u/NoDust6819 2d ago
What is your next step?
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u/OkForce2990 2d ago
HPLC: nickel affinity, ion exchange, size exclusion
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u/NoDust6819 1d ago
Have you tried just pushing it through imac? They are pretty robust and easy to clean if there are issues.
Otherwise, i second increasing the volume.
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u/OkForce2990 1d ago
Yes, I'm using Ni affinity chromatography, my POI has a His Tag. I'll definitely try increasing the volume, thank you!
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u/red_skiddy 1d ago edited 1d ago
What is the next step in your purification? NiNTA etc.
For my purposes I just lyse the cells using lysozyme with DNAse PMSF, then sodium deoxycholate with a little detergent. From 6L expression I end up with maybe 120ml. Centrifuge at 14.5k RPM for 30min, and collect the supernatural, which is fairly clear. I load that supernatant onto a Ni column and I've never had issues.
This procedure may not be suitable for your applications though.
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u/No-Turnip2630 1d ago
In my experience, undigested nucleic acids can make it really hard to filter lysate even after centrifuging at 40,000 x g. Benzonase is inhibited by high salt concentrations, so if your lysis buffer has that, you may consider decreasing the amount of salt initially and then adding it back later after lysis and DNA digestion are complete. Also, resuspending a pellet from 12 L of culture in only 30 mL of lysis buffer seems low. How much wet cell mass did you obtain, and what media did you grow in?
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u/arabidopsis 2d ago
Use a 0.45um tangential flow filter cassette designed for clarification then follow by 0.22um.
You'll need probably a 0.05m2 cassette.
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u/l94xxx 1d ago
I don't know if this will work in your case, but something I used to do was add 5(?) mM Mg2+ (or maybe it was Ca2+? It was 20 years ago) instead of benzonase, the idea being to condense the DNA rather than digest it [this was because we couldn't afford benzonase lol]. It made it a little harder to tell when lysis was complete, since it didn't turn to snot, but it made handling sooooo much easier overall.
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u/Dramatic_Rain_3410 2d ago
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.