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cell lysis method - (May/24/2009 )

Hi all,

I am supposed to treat my cells and determine how much of compound is being uptaken by cells. I grow them in a transwell insert, and I have been harvesting my cells by cell scraping in 0.5 ml PBS, and transfer to microfuge tube, which I would then freeze thaw between -80 to 37C for three times, and further sonicate for 15 minutes. Afterward, I would centrifuge them, and take 5 ul for protein content by bradford and use the rest of my lysate for extraction of my compound. The problem is that I have a relatively high variation between well to well, in term of amount of compound per well, and relatively high variation in protein content too, so when I express my compound per mg of protein, the variation becomes compounded..

I am thus wondering whether there would be a better lysis method than what I am currently using, in term of producing the greatest consistency? And I also observe that even after I lyse the cells, I still see cell clumps... which leads me to think that may be not all of the cells are being lysed.

Thank you!

-zienpiggie-

Is the addition of detergent an issue for your compound quantification? If not, i suggest you lyse your cells in RIPA buffer. Its much simpler and faster than your current lysis method!

-madrius1-