The secret? cytosolic proteins adherent macrophages? - (Oct/13/2007 )
Several companies have proprietary solutions that facilitate isolation of proteins from various adherent cell fractions including cytosol. I am working with macrophages. I want to isolate cytosolic proteins from macrophages; Laemmli buffer gives me problems with 'goop' of RNA and DNA. With most protocols I am getting protein degradation regardless of protease inhibitors in my activated macrophages (I've tried from many companies and many combinations; all dissolved within 20 min of lysis). Anyone know of a protocol/solution that will lyse adherent macrophage cell membranes without lysing other membranes to isolate cytosolic proteins or do only the 'proprietary' solutions have the secret?
I have used activated J774 and RAW cells (macrophage cell lines) to produce inducible iNOS protein for drug screening. I sonicate my cells to release the iNOS protien and then do a cytosolic spin to further "concentrate" my protein. This invovles spinning the supernatant at 105,000g for 30 minutes at 4 oC. I use a buffer containing protease inhibitors and always get "active" protein for kinetic assays for drug screening. The buffer is "home made" but is taken from the litreture.
Send me a private message and I will giev you the composition of my lysis buffer