The activity of CYP3A4, CYP3A5, or CYP2C9 in the presence of WT or mutant POR was tested using the fluorogenic substrate BOMCC (Invitrogen Corp, Carlsbad, CA, USA). The purified CYP3A4, CYP3A5, or CYP2C9 (CYPEX, Dundee, Scotland, UK) were used to test the activities of the POR variants using 20 μM BOMCC as substrate. In vitro CYP3A5 assays were performed using a reconstituted liposome system consisting of pure WT/mutant POR, CYP3A5/CYP2C9, and cytochrome b5 at a ratio of 5:1:1. The final assay mixture consisted of 20 µM DLPC (1,2-Dilauroyl-snglycero-3-phosphocholine)/DLPGV (1,2-Dilauroyl-sn-glycero-3-phosphoglycerol) and proteins (100 nM POR: 20 nM CYP2C9: 20 nM b5), 3 mM MgCl2, 20 μM BOMCC in 50 mM Tris-HCl buffer pH 7.4 and the reaction volume was 100 µL. The CYP3A5 reaction was started by the addition of NADPH to the 1mM final concentration, and fluorescence was measured on a Spectramax M2e plate reader (Molecular Devices, Sunnyvale, CA, USA) at an excitation wavelength of 415 nm and an emission wavelength of 460 nm for BOMCC [21 (link),31 (link),33 (link)].
Bomcc
Manufactured by Molecular Devices
Sourced in United States
BOMCC is a fluorescent dye compound that can be used as a substrate for enzyme activity assays. It emits a measurable fluorescent signal upon enzymatic cleavage, allowing for the quantification of enzyme activity. The core function of BOMCC is to serve as a fluorogenic reporter molecule in biochemical and cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Bomcc, by Thermo Fisher Scientific (5 mentions)
Spectramax m2e, by Molecular Devices (4 mentions)
Cyp2c9, by Thermo Fisher Scientific (2 mentions)
Cyp3a5, by Thermo Fisher Scientific (2 mentions)
Cyp3a5, by Molecular Devices (2 mentions)
Spectramax m2e plate reader, by Molecular Devices (1 mentions)
5 protocols using bomcc
1
Evaluation of CYP Enzyme Activity
2
CYP2C9 Activity Assay with POR Variants
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The activity of CYP2C9 promoted by WT or mutant POR was tested using the fluorogenic substrate BOMCC (Invitrogen Corp, Carlsbad, CA, USA). The purified CYP2C9 (CYPEX, Dundee, Scotland, United Kingdom) was used to test the activities of the POR variants using 20 µM BOMCC as substrate. In vitro CYP2C9 assays were performed using a reconstituted liposome system consisting of WT/mutant POR, CYP2C9, and cytochrome b5 at a ratio of 5:1:1. The final assay mixture consisted of 5 µg DLPC (1,2-Dilauroyl-sn-glycero-3-phosphocholine) and proteins (1 µM POR: 200 nM CYP2C9: 200 nM b5), 3 mM MgCl2, 20 µM BOMCC in 100 mM Tris-HCl buffer PH 7.4, and the reaction volume was 100 µL. The P450 reaction was started by addition of NADPH to a final concentration of 1 mM, and fluorescence was measured on a Spectramax M2e plate reader (Molecular Devices, Sunnyvale, CA, USA) at an excitation wavelength of 415 nm and an emission wavelength of 460 nm for BOMCC.
3
CYP3A5 Activity Assay with POR Variants
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The activity of CYP3A5 promoted by WT or mutant POR was tested using the fluorogenic substrate BOMCC (Invitrogen Corp, Carlsbad, CA, USA). The purified CYP3A5 (CYPEX, Dundee, Scotland, UK) was used to test the activities of the POR variants using 20 µM BOMCC as substrate. In vitro CYP3A5 assays were performed using a reconstituted liposome system consisting of WT/mutant POR, CYP3A5 and cytochrome b5 at a ratio of 5:1:1. The final assay mixture consisted of 5 µg DLPC (1,2-Dilauroyl-sn-glycero-3-phosphocholine) and proteins (1 µM POR: 200 nM CYP2C9: 200 nM b5), 3 mM MgCl2, 20 µM BOMCC in 100 mM Tris-HCl buffer PH 7.4, and the reaction volume was 100 µL. The CYP3A5 reaction was started by addition of NADPH to 1 mM final concentration, and fluorescence was measured on a Spectramax M2e plate reader (Molecular Devices, Sunnyvale, CA, USA) at an excitation wavelength of 415 nm and an emission wavelength of 460 nm for BOMCC.
4
CYP2C9 Activity Assay with POR Variants
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The activity of CYP2C9 promoted by WT or mutant POR was tested using the fluorogenic substrate BOMCC (Invitrogen Corp, Carlsbad, CA, United States). The purified CYP2C9 (CYPEX, Dundee, Scotland, United Kingdom) was used to test the activities of the POR variants using 20 µM BOMCC as substrate. In vitro CYP2C9 assays were performed using a reconstituted liposome system consisting of WT/mutant POR, CYP2C9 and cytochrome b5 at a ratio of 5:1:1. The final assay mixture consisted of 5 µg DLPC (1,2-Dilauroyl-sn-glycero-3-phosphocholine) and proteins (1 µM POR: 200 nM CYP2C9: 200 nM b5), 3 mM MgCl2, 20 µM BOMCC in 100 mM Tris-HCl buffer pH 7.4 and the reaction volume was 100 µL. The P450 reaction was started by addition of NADPH to a final concentration of 1 mM, and fluorescence was measured on a Spectramax M2e plate reader (Molecular Devices, Sunnyvale, CA, United States) at an excitation wavelength of 415 nm and an emission wavelength of 460 nm for BOMCC.
5
Evaluating CYP3A5 Activity with POR Variants
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The activity of CYP3A5 promoted by WT or mutant POR was tested using the fluorogenic substrate BOMCC (Invitrogen Corp, Carlsbad, CA, United States). The purified CYP3A5 (CYPEX, Dundee, Scotland, United Kingdom) was used to test the activities of the POR variants using 20 µM BOMCC as substrate. In vitro CYP3A5 assays were performed using a reconstituted liposome system consisting of WT/mutant POR, CYP3A5 and cytochrome b5 at a ratio of 5:1:1. The final assay mixture consisted of 5 µg DLPC (1,2-Dilauroyl-sn-glycero-3-phosphocholine) and proteins (1 µM POR: 200 nM CYP2C9: 200 nM b5), 3 mM MgCl2, 20 µM BOMCC in 100 mM Tris-HCl buffer pH 7.4 and the reaction volume was 100 µL. The CYP3A5 reaction was started by addition of NADPH to 1 mM final concentration, and fluorescence was measured on a Spectramax M2e plate reader (Molecular Devices, Sunnyvale, CA, United States) at an excitation wavelength of 415 nm and an emission wavelength of 460 nm for BOMCC.
Protein structure analysis of POR variants: Three-dimensional structural models of POR (NCBI# NP_000932) proteins were obtained from the protein structure database (www.rcsb.org). We used the structures of the human POR (PDB # 3QE2) to analyze the
Protein structure analysis of POR variants: Three-dimensional structural models of POR (NCBI# NP_000932) proteins were obtained from the protein structure database (www.rcsb.org). We used the structures of the human POR (PDB # 3QE2) to analyze the
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