I'm looking for a method that will lyse bacterial cells, preferably with minimal use of chemicals and preservation of outer membrane proteins. The gold standard is a "french press", which disrupts cells by subjecting them to rapid extremes in pressure. Sonication with a bit of lysozyme is another possibility. However, I don't have a french press, sonicator probe, or lysozyme. I do, however, have a high pressure ISCO pump and stainless steel mesh of various pore sizes ranging from 10 nm to 100 nm. If I force cells through the mesh, will I get a product similar to what the french press would produce?
Maybe freeze/thaw would be good for you? It requires no special equipment or additives to the bacteria. Here is a link with more information: https://www.thermofisher.com/ch/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/traditional-methods-cell-lysis.html
Hi Kyle,
While I can't speak to your forced-filter plan as I've never tried it, I can propose another possible alternative. In our lab, we regularly use cryo-grinding to perform extremely rapid, non-chemical lysis of cells. Briefly, this involves flash-freezing cells in liquid nitrogen then using a planetary ball mill at -196 Celcius to grind the cells into an extremely fine (sub-cellular level) powder, which we can then use for downstream applications. We've used this technique on several different biological samples (yeast, human/mouse cell culture, cyanobacteria...); however, it does need a planetary ball mill, so I don't know if you have one in your department.
What are you planning to do with your bacterial samples? If you've got a large number of samples and/or are interested in downstream proteomic applications, send me a message - we're a Montreal-based lab (so, not too far from you) and have integrated the above cryo-grinding into a number of rapid, sensitive proteomic/transcriptomic protocols, so could always discuss approaches and/or collaboration.
Hope that helps,
Dan
Thanks for all the input so far. Freeze thaw would be simple and is something I could try, but grinding would not be possible without a planetary ball mill. Another possibility that I like better than force filtering is to fit my ultrasonic drill with a titanium tip of the same diameter as you'd find in an ultrasonic cellular disruptor. Commercial products output the same frequency of 20 KHz, and operate at the same power as my drill. I may even be able to borrow a bit of lysozyme from a colleague.
My goal is to lyse the cells while causing minimal damage to the proteins. This is a control experiment to test under worst case scenarios whether proteins released from dead cells are reacting with something in my flow cell. While it's not critical to preserve the activity of the proteins in my media once they're released (since this wouldn't reflect experimental conditions), I don't want the method of lysis to destroy or change the activity of the proteins.
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