cell lysis method - (May/24/2009 )
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.
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!