We have developed an easy and sensitive method to measure P-glycoprotein (P-gp) activity using [γ-32P]ATP and charcoal. This method utilizes the nature of adsorption of organic phosphate to charcoal. The standard assay method is as follows: the reaction mixture (20 μl) of 1 mM [γ-32P]ATP (1 Ci/mol), 2.5 μg P-gp membranes, and the drug was incubated for 30 min, and 50 μl of 10% charcoal suspension in 0.1 M phosphate buffer at pH7.3 was then added to the mixture. The solution was centrifuged and the supernatant (20 μl) in duplicate containing inorganic 32P was spotted onto filter paper, and radioactivity was measured by radio-image analyzer. This method can reduce the amount of P-gp membrane compared to the conventional method utilizing coloring of the inorganic phosphate-molybdate reaction. This method is also applicable to other ATP-binding cassette (ABC) transporters in phosphate buffer.
Keywords: MDR1, ABCB1, P-glycoprotein, ATP, charcoal, 32P
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Published on: 01 March, 2012
Page: [306 - 310]