TECHNICAL BULLETIN

P450-Glo™ Assays All technical literature is available at: www.promega.com/protocols/ Visit the web site to verify that you are using the most current version of this Technical Bulletin. E-mail Promega Technical Services if you have questions on use of this system: [email protected]

P450-Glo™ Substrate (proluciferin) CYP Enzyme Luciferin Detection Reagent Figure 1. Conversion of P450-Glo™ substrate by cytochrome P450. CYP enzymes act on a luminogenic P450-Glo™ substrate (Reaction A) to produce a luciferin product that generates light with the Luciferin Detection Reagent (Reaction B), which is added after the CYP reaction has been completed. Cytochrome P450 substrate selectivity depends on the specific structure of the proluciferin substrate (Table 1). R varies as shown in Table 1. 2 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in...

luminogenic cytochrome P450 assay in a white plate Luciferin Detection Reagent Reconstitution Buffer or Reconstitution Buffer with esterase reconstituted Luciferin Detection Reagent Figure 2. Flow diagram showing preparation and use of the reconstituted Luciferin Detection Reagent. Use the Reconstitution Buffer provided to reconstitute the lyophilized Luciferin Detection Reagent. Reconstitution Buffer for CYP2C19, CYP2D6 and CYP3A4/Luciferin-IPA assays contains esterase, and the Reconstitution Buffer for all other CYP assays does not contain esterase. Label the blank space on the Luciferin Detection...

(Biochemical Assays) Assays OH 1The arrow indicates the site of modification by CYP. 2The indicated substrate is provided with kits for the CYP shown in bold. These kits also can be used to assay the other CYP enzymes listed for a given substrate. 3The product of the CYP1A2/Luciferin-1A2 and CYP2B6/Luciferin-2B6 reactions is a luciferin precursor. D-luciferin is formed and detected by the Luciferin Detection Reagent supplemented with d-Cysteine. 4CYP2B6 activity was diminished when reactions were supplemented with cytochrome b5. (continued, next page) 4 Promega Corporation ■ 2800 Woods Hollow...

Table 1. Cytochrome P450 Enzymes, Recommended Substrates and Assay Formats (continued). (Biochemical Assays) Assays Luciferin-IPA 1The arrow indicates the site of modification by CYP. 2The indicated substrate is provided with kits for the CYP shown in bold. These kits also can be used to assay the other CYP enzymes listed for a given substrate. Notes for Table 1 Four distinct substrates are available for the CYP3A enzymes. Luciferin-IPA is the most sensitive and selective substrate for all CYP3A4 applications (2), including cell-free or cell-based inhibition assays and cell-based induction...

Description (continued) Notes for Table 1 (continued) Luciferin-PPXE cross-reacts with CYP3A4, 3A5 and 3A7 (Figure 6). The CYP3A4 reaction with Luciferin-PPXE is highly insensitive to DMSO, with little or no inhibition at or below 0.25% DMSO. Luciferin-PFBE is useful for cell-based CYP3A assays. For cell-free enzyme assays Luciferin-PFBE differs from Luciferin-BE in that Luciferin-PFBE is nonreactive with CYP4F12, background luminescence is typically lower than that with Luciferin-BE and its reaction with CYP3A4 is less sensitive to inhibition by DMSO than Luciferin-BE. Luciferin-BE is the original...

2. Product Components and Storage Conditions PRODUCT SIZE CAT.# Each system contains sufficient reagents for 200 biochemical assays at 50pl per assay in 96-well plates. Includes: Each system contains sufficient reagents for 1,000 biochemical assays at 50pl per assay in 96-well plates. Includes: • 1 x 1ml Luciferin-ME, 5mM • 1 vial Luciferin Detection Reagent (lyophilized) Promega Corporation ■ 2800 Woods Hollow Road ■ Madison, WI 53711-5399 USA ■ Toll Free in USA 800-356-9526 ■ 608-274-4330 ■ Fax 608-277-2516 7

2. Product Components and Storage Conditions (continued) PRODUCT SIZE CAT.# Each system contains sufficient reagents for 200 biochemical assays at 50pl per assay in 96-well plates. Includes: Each system contains sufficient reagents for 1,000 biochemical assays at 50pl per assay in 96-well plates. Includes: • 1 x 350pl Luciferin-CEE, 5mM • 1 vial Luciferin Detection Reagent (lyophilized) 8 Promega Corporation ■ 2800 Woods Hollow Road ■ Madison, WI 53711-5399 USA ■ Toll Free in USA 800-356-9526 ■ 608-274-4330 ■ Fax 608-277-2516 TB325 ■ Revised 9/16

PRODUCT SIZE CAT.# Each system contains sufficient reagents for 200 biochemical assays at 50pl per assay in 96-well plates. Includes: Each system contains sufficient reagents for 1,000 biochemical assays at 50pl per assay in 96-well plates. Includes: • 2 x 750pl Luciferin-ME, 5mM • 1 vial Luciferin Detection Reagent (lyophilized) Promega Corporation ■ 2800 Woods Hollow Road ■ Madison, WI 53711-5399 USA ■ Toll Free in USA 800-356-9526 ■ 608-274-4330 ■ Fax 608-277-2516 9 www.promega.com T

2. Product Components and Storage Conditions (continued) PRODUCT SIZE CAT.# Each system contains sufficient reagents for 200 biochemical assays at 50pl per assay in 96-well plates. Includes: Each system contains sufficient reagents for 1,000 biochemical assays at 50pl per assay in 96-well plates. Includes: • 2 x 123pg Luciferin-H EGE • 1 vial Luciferin Detection Reagent (lyophilized) • 1 x 50ml Reconstitution Buffer with esterase 10 Promega Corporation ■ 2800 Woods Hollow Road ■ Madison, WI 53711-5399 USA ■ Toll Free in USA 800-356-9526 ■ 608-274-4330 ■ Fax 608-277-2516 TB325 ■ Revised...

PRODUCT SIZE CAT.# Each system contains sufficient reagents for 200 biochemical assays at 50pl per assay in 96-well plates. Includes: Each system contains sufficient reagents for 1,000 biochemical assays at 50pl per assay in 96-well plates. Includes: • 1 x 60pl Luciferin-IPA, 3mM • 1 vial Luciferin Detection Reagent (lyophilized) • 1 x 50ml Reconstitution Buffer with esterase Promega Corporation ■ 2800 Woods Hollow Road ■ Madison, WI 53711-5399 USA ■ Toll Free in USA 800-356-9526 ■ 608-274-4330 ■ Fax 608-277-2516 11