Hi everyone, I am doing protein expression via E.coli. I am required to lyse the e.coli cells via sonication to obtain cell lysate.
I normally sonicate 1L cell pellet suspended in salt buffer at amplitude:60 hz, 2s on, 2s off, 10 min (x4). 2 minute rest in between. Centrifuge at 13,000 g. Cell suspension appear more translucent after sonication.
After sonicating 5 L pellet, I recombine them into two tubes for centrifugation , I was able to obtain clear yellowish supernatant. However, I noticed that the remaining cell pellet is large with a few black dots. I am not sure if my cells are fully lysed.
My question is: To what extent should I sonicate my cell pellet ?
Is there a clear and indicative sign that my cells are fully lysed ?
Am I supposed to sonicate my cell suspension till it becomes transparent ?
Does high degree of lysis result in smaller pellet after centriguation ?
Thank you.
You can dilute the cell suspension in buffer before centrifugation and look at it under a microscope under high magnification to see if there are a lot of intact bacterial cells.
If you are getting a large pellet after centrifugation at 13,000 g, you probably have not succeeded in getting full cell lysis. There should be a pellet, but it should be much smaller than the original cell pellet.
The black bits are just dust and particles of metal coming off the sonicator probe, not bacteria.
as long as your POI (protein of interest) is easily separated from hen egg white lysozyme its a wonder full addition prior to sonication, and if you throw in a freeze thaw cycle it works better still. Is the POI soluble in lysates or already purified/concentrated in inclusion bodies (IBs)? I've had wonderful results by including sub solubilizing levels of urea and detergent in the sonication mix for insoluble POI as well; the IBs generated were >80% POI. Good luck!
Adam B Shapiro thank you, if the cells are fully lysed, will the pellet after centrifugation still looked the same but smaller ?
I have never seen a fully lysed e.coli pellet before and I can’t find them on google either.
Edward Michelini thank you. My POI is soluble in lysate. I am trying to collect the lysate for fplc purification.
If the cells are fully lysed, the 13,000 g pellet should be very small for a soluble overexpressed protein, although the size will depend on the length of time of the spin. The pellet will consist of cell membranes/cell wall. A longer and/or faster spin will increase the yield of membranes, increasing the size of the pellet, but it will never approach the size of the original cell pellet.
An aliquot was taken every 3-5 min suspension sound and measure the absorbance at 660 nm after dilution. When the A660 stops dropping, you can stop processing.
Hi everyone. Thank you for the response. I recollected the remaining pellet after centrifugation and send it for another cycle of sonication and centrifugation.
I realized that after another round of sonication and centrifugation, the pellet became less thick and more translucent.
I also noticed that there is a colour gradient in the cell pellet with the outer part being yellowish and the inner part being beige in colour. There is also a transparent layer coating the cell pellet.
I have a few more questions:
1. Im just wondering if the different colour represents different component of the cell ?
2. I also noticed that it takes longer centrifugation time (15,000 g, 30-40min) to obtain a clear supernatant. Not sure if it is due to an increase in cell debris or it is due to the small volume of buffer (40ml) used to resuspend the cell pellet.
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