Some protocols suggest heating, others cooling. Should the lysis buffer be ice cold? Is scraping the bottom of the plate necessary?
One protocol suggests using Laemmli buffer in place of lysis buffer to eliminate the spin down step and thereby keep nuclear extracts in the lysate. Is this valid?
What lysis protocol have you found to work best for a western? Specifically, what is most important in terms of temperature, timing, and buffers to get a good lysate?
I am conducting westerns on hepatocytes of human origin and trying to detect a protein between 20 and 30 kD.
It is good to use either clarified cell lysates or purified protein samples for detection using western blot.
Hi,
We do pipetting instead of scraping to detach cells from plate unless your cells are hard to detach. Always keep your sample from this stage on ice. We are using self made 0.5% NP-40 lysis buffer (you may buy a lysis buffer depending your cell line as well). Lysis buffer should be ice cold to prevent protein degradation. After lysis, keep your sample in lysis buffer or in 2XLaemmli buffer at -80 degree C until ready for western blot. Best wishes!
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