is added to the mixture to a concentration of 20 v v and the precipitate removed by centrifugation at 16 000 u00d7 g for 10 minutes additional ethanol is added to the supernatant to a final concentration of 45 and the precipitate constituting the direct cell extract is collected by centrifugation the second stage of the proteosome preparation consists of separating the outer membrane proteins from other membrane components by dissolving either of the products from the first stage the vesicles or the direct cell extract at a concentration of approximately 2 mg protein ml in a buffer hereafter referred to as teen 1 the proteins were then precipitated three times by addition of solid ammonium sulfate at 500 g l of protein solution the precipitates were collected by centrifugation at 30 000 u00d7 g for 20 minutes and redissolved at about 2 mg protein ml in teen 1 the final precipitate was dissolved with the aid of a water bath sonicator at about 2 mg ml centrifuged at 16 000 u00d7 g for 20 minutes to remove insoluble material and then dialyzed against tween 0 1 to remove any residual ammonium sulfate the final concentration of empigen bb can be 0 1 to 1 0 products are stored at u2212 20 u00b0 c or for short periods at 4 u00b0 c the proteosomes prepared from bacteria other than those prepared from meningococci may also be prepared and used by the same methodology in certain instances the hydrophobic foot attached through.