UGT Activity Assay / Ligand Screening Kit (Fluorometric) (ab273331)
Key features and details
- Assay type: Enzyme activity (quantitative)
- Detection method: Fluorescent
- Platform: Microplate
- Sample type: Adherent cells, Microsomes, Suspension cells, Tissue
- Sensitivity: 0.1 mU/well
Overview
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Product name
UGT Activity Assay / Ligand Screening Kit (Fluorometric) -
Detection method
Fluorescent -
Sample type
Tissue, Adherent cells, Suspension cells, Microsomes -
Assay type
Enzyme activity (quantitative) -
Sensitivity
0.1 mU/well -
Assay duration
Multiple steps standard assay -
Product overview
UGT Activity Assay / Ligand Screening Kit (Fluorometric) (ab273331) enables rapid measurement of native or recombinant UGT activity in biological samples such as liver microsomes and can also be used to assess the effect of drugs and other novel compounds on UGT activity. The assay utilizes a highly fluorescent UGT substrate with a large Stokes shift (Ex/Em = 415/502 nm) that allows determination of UGT activity by tracking the drop in fluorescence emission as the substrate is converted into a non-fluorescent glucuronide. The multi-isozyme substrate is glucuronidated by virtually all of the pharmacologically-relevant mammalian UGT1A and UGT2B enzymes. UGT specific activity is calculated by comparing the fluorescence loss versus a control reaction performed in the absence of the required cofactor UDPGA. The kit includes the pore-forming peptide antibiotic Alamethicin, which allows the UGT Substrate and UDPGA to rapidly diffuse across lipid membranes to access the UGT active site located in the lumen of microsomes. For verification of modulation of UGT activity by test ligands, diclofenac, a competitive inhibitor of most human and rodent UGT isozymes, is also included. The assay is highly sensitive, simple to perform and high-throughput adaptable. This assay can detect less than 0.1 mU UGT activity in biological samples. The kit contains a complete set of reagents sufficient for performing 100 reactions at a 100 μl reaction volume.
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Platform
Microplate
Properties
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Storage instructions
Store at -20°C. Please refer to protocols. -
Components 100 tests Alamethicin 1 x 50µl UDPGA Stock (50X)(Lyophilized) 1 vial UGT Assay Buffer 1 x 100ml UGT Inhibitor (Diclofenac)(Lyophilized) 1 vial UGT Positive Control (Lyophilized) 1 vial UGT Substrate (250X)(Lyophilized) 1 vial -
Research areas
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Cellular localization
Microsome membrane; Single-pass membrane protein. Endoplasmic reticulum membrane; Single-pass membrane protein -
Alternative names
- Bilirubin-specific UDPGT isozyme 1
- Hyodeoxycholic acid-specific UDPGT
- UDP-glucuronosyltransferase 1-1
see all
Images
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UGT Substrate Standard Curve.
Typical standard curve – data provided for demonstration purposes only. A new standard curve must be generated for each assay performed.
UGT Substrate Standard Curve.
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Reaction KineticsReaction kinetics of fluorescent substrate glucuronidation in donor-pooled human liver microsomes (HLMs, 0.0625 mg/mL) at 37°C and inhibition of UGT activity in HLMs by the isozyme-selective ligands propofol (UGT1A9-selective) and zidovudine (UGT2B7-selective), as well as the non-selective UGT ligand diclofenac. For the blank reaction condition, vehicle (Assay Buffer) was substituted for the cofactor UDPGA.
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Specific Activity in MicrosomesSpecific UGT activity in pooled human and rat liver microsomes (mean ± SEM of 3 replicates).
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Dose-response curve for UGT inhibition by diclofenac in HLMs.Dose-response curve for UGT inhibition by diclofenac in HLMs. Percent activity was calculated for each concentration by comparison to activity of reaction containing vehicle only. IC50 values were derived by 4-parameter logistic curve fitting with each point representing the mean ± SEM of at least 3 replicates. Assays were performed according to the kit protocol.