I do an 8L induction at 18C for 16-20 hours in TB media. I routinely harvest ~50-80g of bacteria. I freeze my pellets at -20 for > 3 hours after harvesting, and have been using a 1kW blender to resuspend and homogenize them in my lysis buffer. However, I have been noticing that the blender causes a lot of air to be entrained in my lysate, which seems to be affecting my protein yield. Is there a better method to resuspend and homogenize my quantity of bacteria?
I can provide more information upon request.
The trick is to resuspend THEN lyse, not a combination! I always diluted my induced cells and pelleted at a set mass, say 20-50g per tube/bottle then snapped freeze them in dry ice methanol bath and stored at -80C. For long term storage I'd dip bottles in hot water just until frozen pellet cracked off bottle wall and transferred still frozen pellet to plastic bags for continued -80C storage. When I wanted to isolate proteins I'd thaw in PBS at 4C with gentle shaking. When entire pellet was resuspended I'd hit with lysis buffer which always included protease inhibitor cocktail and lysozyme, then probe sonication. I was isolating IBs so I then did repeated washes/pelleting with fresh urea/surfactants and sonication, at levels under what dissolved IBs, then solubilize when pellets were almost pure product and refold. Good luck!
If your lysis buffer contains detergent, foaming of the suspension during blending is inevitable. Since detergent is not necessary for homogenization, only for lysis, and the blender is used only for homogenization, my suggestion is to use a blending buffer that lacks detergent, then add the detergent afterwards to perform the lysis. Keeping the blender to the minimum speed should also help avoid incorporating a lot of air.
Break up your pellets into smaller chunks using a spatula and then use a magnetic stirrer. This will take longer than the blender but should introduce less air.
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