Protein extraction - (May/17/2012 )
I am trying to extract proteins from MCF7 and BT20 cell lines in order to analyze through a western blot. The protein of interest is Cyp1b1 which is Flag tagged. Since Cyp1b1 is bound to the smooth ER, I purchased a Pierce Protein Extraction Kit (http://www.piercenet...CFQSynQodDRNGLw).
Since I am going to be looking at protein degradation at different time points (cyclohexamide treatment and protein extracted 0hrs, 4hrs, and 8hrs), I was told that I would need protease inhibitors to keep my protein from being degraded after extraction. This is the first time my lab has worked with human cell lines, and nobody is 100% sure of what all that entails. I hope somebody on here can help me out a bit as to what I need to purchase in order to extract the protein which will be used for westerns.
Thanks in advance,
Typically you would make what is known as a lysate - this is basically dissolved cells in a solution that contains detergent(s) (often NP-40, nonidet-p40, triton and/or sds), buffer (usually tris) and salt (50-300 mM). To this is added protease inhibitors to prevent the proteins being degraded, and if necessary, phosphatase inhibitors.
Protease inhibitors are usually a combination of NaF, Na3VO4, and PMSF. You can buy these individually, but I would recommend a combined protease inhibitor cocktail such as Complete from Roche, or any of the ones from Sigma Aldrich, as they are notoriously unstable. These mixs conveniently contain all the inhibition you need in one tablet or solution in stabilised form.
Have a look for any one of the many RIPA buffers for a generalized protein solubilisation buffer.
naf and na3vo4 are phosphatase inhibitors.
sigma protease inhibitor cocktail includes: AEBSF, Bestatin, E-64, Pepstatin A, Phosphoramidon, Leupeptin, with or without edta
Thanks for the help! I really appreciate it!
sigma protease inhibitor cocktail is more efficient than other protease inhibitor cocktails.