Corning® Gentest™ - Corning Incorporated

** Use 1.5 mM and 0.5 mM solutions to generate the last two points on the curve.0 - 0 400 00.05 1.5 ** 40 360 30.15 1.5 ** 40 360 90.5 10 20 380 301.0 10 40 360 601.5 10 60 340 902.0 10 80 320 1202.5 10 100 300 150Final Phosphate in WellPhosphate Phosphate Stock Volume of Phosphate Volume of (After 60 µL addition)Standard (mM) to be Used (mM) Stock (µL) Assay Buffer (µL) (nanomoles)Phosphate Standard CurveTo place an order in the U.S.Contact BD Biosciences Customer Support at:tel: 877.232.8995, fax: 800.325.9637 or 858.812.8889;online: shop.bdbiosciences.com.For technical assistance, contact Technical Support at:tel: 877.232.8995 or 978.901.7389; fax: 978.901.7491;e-mail: labware@bd.comOutside the U.S.Contact your local distributor or visit bdbiosciences.com/offices to locate your nearest BD Biosciences office.Corning acquired the Discovery Labware Businessincluding the Gentest brand. For additional Corningproduct, technical, or distributor information, pleasee-mail us at CLSTechServ@corning.com, visit our websitewww.corning.com/lifesciences or call 800.492.1110. Outsidethe United States, call 978.442.2200. For information onthe acquisition, visit www.corning.com/discoverylabware.Corning IncorporatedLife Sciences836 North St.Building 300, Suite 3401Tewksbury, MA 01876t 800.492.1110t 978.442.2200f 978.442.2476www.corning.com/lifesciencesFor Research Use Only. Not for usein diagnostic or therapeutic procedures.For a listing of trademarks, visit us atwww.corning.com/lifesciences/trademarks.Corning Incorporated, One Riverfront Plaza,Corning, NY 14831-0001© 2012, 2013 Corning Incorporated Printed in USA 2/13 CLS-DL-GT-018Corning ® Gentest ABC Transporter MembraneATPase AssayQUICK START GUIDERefer to Kit Instruction Manual for detailed procedure.

Corning® Gentest ABC Transporter Membrane ATPase AssayExample for human P-gp K mAssay for one compoundCorning® 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