Corning® Gentest⢠- Corning Incorporated
Corning® Gentest⢠- Corning Incorporated
Corning® Gentest⢠- Corning Incorporated
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<strong>Corning</strong>® Gentest ABC Transporter Membrane ATPase AssayExample for human P-gp K mAssay for one compound<strong>Corning</strong>® GentestABC Transporter MembranesSAMPLE 1SAMPLE 2IncubationDescription Cat. No. Substrate Time (minutes)5 mg/mL P-gp membraneH 2OAssay buffer90 µL13.5 µL346.5 µL5 mg/mL P-gp membrane10mM NaOVAssay buffer90 µL13.5 µL346.5 µLPREPARATION OFSTANDARD CURVEHuman P-gp(MDR1) Membranes 453228 Verapamil 20Cynomolgus MonkeyP-gp Membranes 453230 Verapamil 30Aliquot 20 µLAliquot 20 µLAliquot 60 µLRhesus MonkeyP-gp Membranes 453231 Verapamil 30COLUMNS 3-12ROWS A & BABSTANDARD(mM)CTRL SERIAL DILUTIONS OF TEST COMPOUNDS (µM)1 2 3 4 5 6 7 8 9 10 11 12150 150 Ctrl 1 300120 120 Ctrl 1 300150 75 37.5150 75 37.518.75 9.375 4.68 2.34 1.1718.75 9.375 4.68 2.34 1.17Testcompound 1COLUMNS 3-12ROWS C & DSTANDARDCOLUMNS 1 & 2ROWS A-HBeagle DogP-gp Membranes 453229 Verapamil 30Rat Multi-Drug-Resistance1a (Mdr1a) Membranes 453440 Verapamil 30ASSAY SAMPLEPREPARATIONCDEFGH906030930906030930Ctrl 2Ctrl 2Ctrl 1Ctrl 1Ctrl 2Ctrl 230030030030030030015015015015015015075757575757537.537.537.537.537.537.518.7518.7518.7518.7518.7518.759.3759.3759.3759.3759.3759.3754.684.684.684.684.684.682.342.342.342.342.342.341.171.171.171.171.171.17Testcompound 1+NaOVTestcompound 2Testcompound 2+NaOVRat Multi-Drug-Resistance1b (Mdr1b) Membranes 453434 Verapamil 30Mouse Multi-Drug-Resistance1a (Mdr1a) Membranes 453232 Verapamil 30Mouse Multi-Drug-Resistance1b (Mdr1b) Membranes 453250 Verapamil 30Add 20 µL of serial diluted test compoundHuman MRP1 Membranes 453456 NEM/GSH 60Preincubate at 37 o C for 5 minutesRat Mrp1 Membranes 453340 NEM/GSH 40ASSAY INCUBATIONAdd 20 µL of ATP into columns 3-12, row A-DHuman MRP2 Membranes 453332 Probenecid 40Incubate at 37 o C for desired amount of timeRat Mrp2 Membranes 453334 Probenecid 40STOP ASSAYAdd 30 µL of Assay Stop Solution into all wellsPrepare colorimetric reagentHuman MRP3 Membranes 453333 Benzbromarone 60Human BCRP(Arg482) Membranes 453270 Sulfasalazine 10COLORIMETRICASSAYAdd 200 µL of colorimetric reagent into all wellsIncubate at 37 o C for 20 minutesMouse Bcrp Membranes 453251 Sulfasalazine 30Rat Bcrp Membranes 453457 Sulfasalazine 10Control Membranes 453200 — —Read the plate on spectrophotometer at wavelength of 800 nm
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