Cytochrome P450 Protocols by Douglas S. Auld, Henrike Veith, James J. Cali (auth.), Ian

By Douglas S. Auld, Henrike Veith, James J. Cali (auth.), Ian R. Phillips, Elizabeth A. Shephard, Paul R. Ortiz de Montellano (eds.)

Cytochromes P450 (CYPs) contain a wide superfamily of proteins which are of critical significance within the cleansing or activation of an incredible variety of normal and artificial hydrophobic xenobiotics, together with many healing medications, chemical cancer agents and environmental toxins. CYPs are very important in mediating interactions among an organism and its chemical surroundings and within the law of physiological techniques. Cytochrome P450 Protocols, 3rd variation focuses on high-throughput tools for the simultaneous research of a number of CYPs, substrates or ligands. even supposing the emphasis is on CYPs of mammalian starting place, it displays an expanding curiosity in CYPs of bacterial species. additionally incorporated are chapters on cytochrome P450 reductase (the redox accomplice of CYPs) and the flavin-containing monooxygenases (FMOs), and metabolomic and lipidomic techniques for identity of endogenous substrates of CYPs (‘de-orphanizing’ CYP substrates). Written within the winning Methods in Molecular Biology™ sequence layout, chapters contain introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, effortlessly reproducible protocols, and notes on troubleshooting and heading off recognized pitfalls.

Authoritative and simply available, Cytochrome P450 Protocols, 3rd version provides quite a lot of ideas available to researchers in fields as diversified as biochemistry, molecular biology, pharmacology, toxicology, environmental biology and genetics.

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12. 6, step 41 above, onto the RapidFire sample stage. 13. ” 14. Click “OK” to start the data acquisition on the mass spectrometer. 15. Click “start” (the button with green triangle symbol) on the RapidFire instrument control software to start sample analysis on the RapidFire (see Note 24). 16. When the analysis for the first plate is finished, swap in the second analysis plate (MS-2) and click “continue” to start the data acquisition for the second plate (see Note 25). 17. Repeat step 16 for additional plates.

2. 4. Make sure the tip boxes containing DITI (TB-#) are loaded (see Note 12) on deck as shown in Fig. 2. 5. Check the Tecan calibration (see Note 13). 6. Start the Tecan program (see Note 14) to run the assay. It will take about 30 min for the entire run. 6. 6 Sequence of Automated Assay 1. MCA arm move to pick up tips from TB-1. 2. Aspirate reagents from ESP-1A2 and sequentially dispense 66 mL into AP-1A2-1 and AP-1A2-2. 3. Discard the used tips. 4. Repeat steps 2–3 for the enzymes/substrates of 5 other isozymes, using TB-2, TB-3, TB-4, TB-5, and TB-6 (see Note 15).

12. Typically, concentrations in the 1–5 mM range are used for single concentration DDI assessment (15, 39). 13. Strict adherence to initial rate and compliance with Michaelis– Menten kinetics (<10% substrate depletion) is not possible with very different reaction rates, although with an 8-min reaction time no more than 25% substrate depletion is observed for the most extensively metabolized substrate while maintaining sensitivity of the least metabolized substrate (20). 20 Michael Zientek and Kuresh Youdim 14.

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