Biochemical Nuclear Receptor Assays - Kits

LanthaScreen™ TR-FRET CAR Coactivator Assay Kit, goat

This kit contains Goat Tb-Anti-GST antibody; other kit components are the same as kit A15141:

The LanthaScreen® TR-FRET Constitutive Androstane Receptor (CAR) Coactivator Assay provides a sensitive and robust method for highthroughput screening (HTS) of potential CAR ligands as agonists of ligand-dependent coactivator recruitment or inverse agonists of liganddependent coactivator displacement. The kit uses a terbium-labeled anti-GST antibody, a fluorescein-labeled coactivator peptide, and a CAR ligand-binding domain (CAR-LBD) that is tagged with glutathione-S-transferase (GST) in a homogeneous mix-and-read assay format.

To assay:
When running the LanthaScreen® TR-FRET CAR Coactivator Assay, CAR-LBD is added to ligand test compounds followed by addition of a mixture of the fluorescein-coactivator peptide and terbium anti-GST antibody. After an incubation period at room temperature, the 520 nm/ 495 nm TR-FRET ratio is calculated and can be used to determine the EC50 from a dose response curve of the compound. Based on the biology of the CAR-coactivator peptide interaction, this ligand’s EC50 is a composite value representing the amount of ligand required to bind to receptor, effect a conformational change, and either recruit or displace coactivator peptide (Figures 1 and 2).

LanthaScreen™ TR-FRET ERR gamma Coactivator Assay Kit, rabbit

This kit contains Rabbit Tb-Anti-GST antibody; other kit components are the same as kit PV4408:

The LanthaScreen® TR-FRET Estrogen Related Receptor (ERR) gamma Coactivator Assay Kit provides a sensitive and robust method for high-throughput screening of potential ERR gamma ligands as antagonists/inverse agonists of coactivator displacement. The homogeneous mix-and-read assay format results in a ligand EC50 composite value representing the amount of ligand required to bind to receptor, effect a conformational change, and displace coactivator peptide (Figure 1).

How it works

The LanthaScreen® TR-FRET ERR gamma Coactivator Assay Kit includes a terbium (Tb)-labeled anti-GST antibody, a fluorescein-labeled coactivator peptide, and a human ERR gamma ligand-binding domain (ERR gamma-LBD) that is tagged with glutathione-S-transferase (GST). To assay, ERR gamma-LBD is added to test compounds, followed by addition of a mixture of the fluorescein-coactivator peptide and Tb-anti- GST antibody. After room temperature incubation, the TR-FRET 520:495 emission ratio is calculated and used to determine the EC50 from a dose response curve of the compound.

Contents and Storage:

The LanthaScreen® TR-FRET ERR gamma Coactivator Assay Kit contains ERR gamma-LBD (GST) protein, fluorescently labeled PGC1a coactivator peptide, Tb-anti-GST antibody, and buffers. Store components as indicated in the assay protocol (-80°C, -20°C, or +4°C).

LanthaScreen™ TR-FRET ER beta Coactivator Assay Kit

The LanthaScreen® TR-FRET ER beta Coactivator Assay Kit provides a sensitive and robust method for high-throughput screening of potential estrogen receptor (ER) beta ligands as agonists or antagonists of ligand-dependent coactivator recruitment. The kit uses a ER beta ligand-binding domain (ER beta-LBD) that is tagged with glutathione-S-transferase (GST), a terbium (Tb)-labeled anti-GST antibody, and a fluorescein-labeled coactivator peptide in a homogenous mix-and-read assay format.

Agonist Mode
To run the LanthaScreen TR-FRET ER beta Coactivator Assay in agonist mode (to identify agonist compounds), ER beta-LBD is added to ligand test compounds followed by the addition of a mixture of fluorescein-coactivator peptide and Tb-anti-GST antibody. After room temperature incubation, the TR-FRET 520:495 emission ratio is calculated and used to determine the EC50 from a dose response curve of the compound. Based upon the biology of the ER beta-coactivator peptide interaction, this ligand EC50 is a composite value representing the amount of ligand required to bind to receptor, effect a conformational change, and recruit coactivator peptide.

Antagonist Mode
When the LanthaScreen TR-FRET ER beta Coactivator Assay Kit is run in antagonist mode (to identify antagonist compounds), ER beta-LBD is added to ligand test compounds followed by the addition of a mixture of agonist, fluorescein-coactivator peptide, and Tb-anti-GST antibody. The concentration of agonist used in this mode is the EC80 concentration as determined by first running the assay in agonist mode.

PolarScreen™ PPARγ-Competitor Assay Kit, Green

The PPARγ (Peroxisome Proliferator-Activated Receptor-gamma) Competitor Assay, Green uses the human-derived recombinant PPARγ ligand-binding domain (PPARγ-LBD) tagged with an N-terminal GST-tag and a novel, tight-binding, selective fluorescent PPARγ ligand (Fluormone™ PPARγ Green) in a homogenous, mix-and-read assay format. PPARγ-LBD/Fluormone™ PPARγ Green complex produces high polarization. This complex is then added to individual test compounds in microwell plates. Competitors displace the fluorescent Fluormone™ PPARγ Green Ligand from the PPARγ-LBD/Fluormone™ PPARγ Green complex, causing the fluorescent ligand to tumble rapidly during its fluorescence lifetime, resulting in a low polarization value. Noncompetitors will not displace the fluorescent ligand from the complex, so the polarization value remains high. The shift in polarization value in the presence of the test compounds is used to determine relative affinity of test compounds for the PPARγ-LBD.

LanthaScreen™ TR-FRET RXR beta Coactivator Assay Kit, goat

This kit contains Goat Tb-Anti-GST antibody; other kit components are the same as kit A15120:

The LanthaScreen® TR-FRET Retinoid X Receptor (RXR) beta Coactivator Assay Kit provides a sensitive and robust method for high-throughput screening of potential RXR beta ligands as agonists of coactivator recruitment. The homogeneous mix-and-read assay format results in a ligand EC50 composite value representing the amount of ligand required to bind to receptor, effect a conformational change, and recruit coactivator peptide (Figure 1).

How it works

The LanthaScreen® TR-FRET RXR beta Coactivator Assay Kit includes a terbium (Tb)-labeled anti-GST antibody, a fluorescein-labeled coactivator peptide, and a human RXR beta ligand-binding domain (RXR beta-LBD) that is tagged with glutathione-S-transferase (GST). To assay, the RXR beta-LBD is added to ligand test compounds, followed by addition of a mixture of the fluorescein-coactivator peptide and Tb-anti-GST antibody. After room temperature incubation, the TR-FRET 520:495 emission ratio is calculated and used to determine the EC50 from a dose response curve of the compound

Contents and Storage:

The LanthaScreen® TR-FRET RXR beta Coactivator Assay Kit contains RXR beta-LBD (GST) protein, fluorescein-labeled D22 coactivator peptide, Tb-anti-GST antibody, and buffers. Store components as indicated in the assay protocol (-80°C, -20°C, or +4°C).

PolarScreen™ ER Beta Competitor Assay Kit, Red

The PolarScreen™ ER Beta Competitor Assay Kit, Red, provides a sensitive and efficient method for high-throughput, fluorescence polarization-based screening of potential estrogen receptor (ER) beta ligands. The kit uses insect cell-expressed, full-length, native (untagged) human ER beta (A15664) and a novel, high-affinity, fluorescent estrogen ligand (Fluormone™ ES2) in a homogenous, mix-and-read assay format.

This kit produces a red color read-out; a "green" kit is also available (A15890). Both kits suitably determine the IC50s of competitive compounds. Color choice depends on the spectral properties of the compounds to be screened and the available instrumentation. For additional information or assistance email drugdiscoverytech@lifetech.com.

The PolarScreen™ ER Beta Competitor Assay Kit is:

• Complete & ready-to-use out of the box—all reagents included, just add your test compounds
• High throughput compatible—20 µL fluorescence polarization assays in a 384-well plate
• Convenient—available in bulk sizes for larger screening needs

How the Assay Works
Full-length ER beta is added to a fluorescent estrogen ligand to form an ER-Fluormone™ ES2 complex. This complex is added to individual test compounds in multiwell plates. To determine relative affinity, the assay measures fluorescence polarization. Competing test compounds will displace the Fluormone™ ES2 ligand from ER beta, permitting it to tumble rapidly and resulting in low polarization values. A shift in the fluorescence polarization value in the presence of test compound is used to determine the relative affinity of test compounds for ER beta.

All components are manufactured under the strictest quality parameters. This product's performance, per the Certificate of Analysis, is guaranteed for 6 months after purchase. Contact us for bulk quantities or for assistance with other nuclear receptor assay needs, from assay development to library screening and beyond.