Lysis buffer - (Nov/07/2012 )
I have found several protocols for adherent cell lysis buffer (NP-40). Some have 10mM,20mM or 50mM Tris HCl. Why is there a diference? Also, it was suggested that RIPA buffer is not compatible with Bio-Rad bradford assay reagent, however in the guidelines of the reagent it is mentioned that up to 0.1% SDS is compatible. Any recommendations for good lysis buffer protocols for both membrane and cytosolic protein extraction?
The tris is a buffering system, the more tris in there, the better your lysis will stay at the pH the tris was at before lysis (usually pH 7.4).
RIPA buffer is pretty standard for most applications. I don't know if it is compatible with the Bradford assay - check the technical documentation that comes with the assay.
The choice of lysis buffer can be based on the subcellular location of your protein(s) of interest. Some detergents solubilize certain proteins better. Abcam gives a short overview: http://www.abcam.com/ps/pdf/protocols/WB-beginner.pdf