Multidrug Efflux Transporter (MDR1/P-gp) Ligand Screening Kit (BN00738)

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BN00738
  • Multidrug Efflux Transporter (MDR1/P-gp) Ligand Screening Kit (BN00738)
  • Multidrug Efflux Transporter (MDR1/P-gp) Ligand Screening Kit (BN00738)
  • Multidrug Efflux Transporter (MDR1/P-gp) Ligand Screening Kit (BN00738)
€505

Description

ELISA Kit Technical ManualMSDS

Multidrug Efflux Transporter (MDR1/P-gp) Ligand Screening Kit

P-glycoprotein (P-gp, Multidrug Resistance Protein 1 (MDR1), EC 3.6.3.44) is a member of the ATP-binding cassette (ABC) ATPase superfamily of transmembrane transporter proteins. P-gp has an extremely broad substrate specificity and is capable of transporting a vast array of neutral and anionic lipophilic molecules. P-gp strongly affects the oral absorption, tissue distribution and excretion of many drugs and prevents certain lipophilic drugs from penetrating the blood brain barrier. Overexpression of P-gp confers tumor cells with resistance to chemically and pharmacologically distinct chemotherapeutic drugs (such as doxorubicin, vincristine and paclitaxel) by actively pumping them out of cells. Induction of P-gp expression is a frequent cause of treatment failure and tumor-targeted delivery of P-gp inhibitors is being investigated as a strategy for overcoming chemotherapy resistance. Assay Genie's MDR1/P-gp Ligand Screening Kit is designed for rapidly screening test compounds for modulation of efflux transporter activity in MDR1-expressing cell lines. The assay uses a lipophilic non-fluorescent P-gp substrate that readily diffuses through the plasma membrane, where it is hydrolyzed to an active fluorophore by cytosolic esterases. The resulting hydrophilic fluorophore is neither membrane permeable nor a substrate for P-gp, hence it remains trapped inside the cell. In MDR1-expressing cell lines, the lipophilic pro-fluorophore is continuously extruded from the cytosol by P-gp, leading to a low intracellular fluorescence. Inhibition of P-gp-mediated efflux by a test compound leads to increased intracellular fluorescence. Specific transporter activity is quantified by comparison with fluorescence accumulated in the presence and absence of a saturating concentration of the included selective P-gp inhibitor. The assay is highly sensitive, has a simple no-wash protocol and is high-throughput adaptable. The kit contains a complete set of reagents sufficient for performing 100 reactions in a 96-well plate format.

Figure: (a) Intracellular accumulation of fluorogenic MDR1 substrate hydrolysis product in the presence and absence of the MDR1 inhibitors verapamil and cyclosporin A. Fluorescence was measured 30 min after addition of MDR1 substrate. (b) Dose-response curves for MDR1 inhibition by verapamil and cyclosporin A. Percent activity was calculated for each concentration by comparison to transporter (a) activity in the presence of 100 μM verapamil (positive inhibition control) and vehicle (negative inhibition control). (c) Fluorescence microscopy showing increased intracellular fluorescence in the presence of verapamil (middle right panel) and cyclosporin A (lower right panel). Cells were cultured overnight on a clear-bottom 96-well plate and exposed to MDR1 substrate for 30 min following 30 min pre- incubation with either vehicle (1% DMSO), verapamil (100 μM) or cyclosporin A (10 μM). Brightfield and fluorescence images were obtained with a Nikon TE2000 inverted microscope using a 10X Plan Fluor objective. All assays were performed according to the kit protocol using MES-SA/MX2 cells (ATCC CRL-2274).

Key Information Description

Product SKU

BN00738

Size

100 Assays

Detection Method

Fluorescence (Ex/Em = 488/532 nm)

Applications

Screening/studying/characterizing MDR1/P-gp Inhibitors

Features and Benefits

  • Simple method to screen Pg-p inhibitors
  • High-throughput adaptable
  • Includes Inhibitor Control, Verapamil

Kit Components

  • Efflux Assay Buffer
  • Fluorogenic P-gp Substrate
  • P-gp Inhibitor (Verapamil)

Storage Conditions

-20°C

Shipping Conditions

Gel Pack

USAGE

For Research Use Only! Not For Use in Humans.

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