Reverse FECH

• Sonicate the washed pellet of cultured bacterial cells in two volumes of 10mM KPi pH 5.5. Determine protein content and adjust to about 20mg/mL with the same buffer to prepare the homogenate.  Deoxygenate all samples and reagents.
• Mix about 50µL homogenate (1.0 mg total protein) with 50 μL of assay reagent containing 100 μM hemin-imidazole, 4 mM ascorbic acid in 10 mM potassium phosphate buffer pH 5.5 under argon atmosphere.
• Cap the sample tubes securely, incubate for 1h at 45˚C in a water bath and then add 400 μL 50% v/v acetone in ethanol.
• Centrifuge the samples at 13,500 g for 10 min.
• Analyze the supernatant for PPIX fluorescence by UPLC (ultra-performance
• liquid chromatography).
• For blank controls samples of bacterial homogenates were heated for 10 min in a boiling water bath and assayed with the live samples