W571:zegzouti:ADPGLOMAX
From LabAutopedia
Detecting ATPase activity modulators of ABC transporters using ADP-Glo Max
Zegzouti Hicham*; Riss Terry and Goueli Said
Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA
To ensure drug candidates will have good bioavailability and distribution and less toxicity, it is important to discern drug interaction with ATP-binding cassette (ABC) transporters early in drug discovery. ABC transporters are trans-membrane proteins with an ATPase activity that is both coupled to transporting activity and stimulated by transported drugs. Therefore, identifying how a drug modulates transporter ATPase activity can lead to a better understanding of the interaction. Because these transporters have a low ATP turnover rate, a sensitive assay is needed and a high throughput format is desirable for early screening efforts. The ADP-Glo™ Max Luminescent ADP detection assay provides a universal, homogeneous, and high-throughput method for measuring ATPase activity by quantifying the amount of ADP produced during the reaction. The assay is sensitive enough to detect low amounts of ADP in the presence of high amounts of ATP for a broad range of ATP concentrations (up to 5mM). This is a distinct advantage for transporters which typically have a high Km for ATP. The luminescent signal generated is proportional to the ADP concentration produced and it correlates to the amount of ATPase activity. Here we present the application of the assay to the identification of substrates and inhibitors of the ABC transporters P-gp, BCRP, MRP1, MRP2, and MRP3. The ADP-Glo™ Max Assay can be used with virtually all ATPases and because of its exquisite sensitivity it is especially useful for transporters with low ATP turnover. Only a small amount of transporter membranes (10µg) was required to achieve a high signal to background ratio. The ADP-Glo™ Max luminescent detection scheme also minimizes interferences from reaction components that interfere with other detection schemes. This highly sensitive readily automatable assay can easily replace the low throughput, significantly less sensitive colorimetric or fluorescent ATPase assays that are based on inorganic phosphate detection.
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