Purification and Analysis of Retinoids

Kanamycin, sodium chloride, potassium chloride, Tris, Hepes, potassium phosphate, glycerol, benzonase, lysozyme, thrombin, protease inhibitor tablets, Coomasssie Brilliant Blue and atRA were purchased from Millipore-Sigma (St. Louis, MO, USA). The 4-oxo-atRA-d₃ was purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and 4-OH-atRA was purchased from Toronto Research Chemicals (North York, ON, Canada). Sodium hydroxide, PMSF, imidazole, Pierce BCA protein assay, DTT, EDTA, IPTG, tryptone, yeast extract, high-performance liquid chromatography (HPLC) and mass spectrometry grade acetonitrile, water, and ethyl acetate were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Lipidex-5000 was purchased from Perkin Elmer Inc (Waltham, MA, USA). Mini-PROTEAN TGX protein gels were purchased from Bio-Rad (Hercules, CA, USA). NBD-stearate was purchased from Avanti Polar Lipids (Birmingham, AL, USA). Pooled human liver microsomes were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Recombinant CYP3A4 and CYP2C8 Supersomes co-expressed with cytochrome P450 reductase and cytochrome b5 were purchased from Corning (Glendale, AZ, USA). Recombinant CYP26A1 was expressed in Sf9 insect cells, as previously described [17 (link),20 (link)]. Rat P450 reductase was expressed in E. coli and purified, as previously described [33 (link)].