at my the end of my rope trying to purify this protein. So I recently started a postdoc and have been tasked with purifying a microsomal P450 enzyme. I did purification of his tagged heme proteins during my PhD and ever had any trouble. This P450 will not stick to the column no matter what I do. Here is a brief explanation of my protocol and what I have tried. I grow the cells in TB media and induce with. 1 mM IPTG and 1 mM 5-ALA and let the expression go on for 48 hours at 25 C. I collect the cell pellet and lyse in a buffer composed of 50 mM tris HCl pH 7.4 with 150 mM NaCl, 20% glycerol, 0.4% triton x 100 or 1% CHAPS (I’ve tried both), 1 mM PMSF, 0.5 mM DTT, 10 mM imidazole, and an EDTA free protease inhibitor cocktail. I then centrifuge at 21000g for about an hour to collect the membrane debris. I load this onto the column using an Akta fplc with a flow rate of 0.5 mL per minute (25 mL column). My wash buffers are 50 mM tris HCl pH 7.4, 20% glycerol, 0.4% triton X 100 or 1% CHAPS, 250 mM NaCl.…