| Assay Method Information | |
| | In vitro ATRA 4-Hydroxylase Activity Assay |
| Description: | All procedures were carried out under minimal light in order to prevent degradation of the retinoid samples.Microsomal preparation: one lobe of fresh pig liver was obtained at the time of slaughter from a food-processing company and immediately placed in ice cold 15 mM KH2PO4/250 mM sucrose (pH 7.4) and kept on ice during transportation. A 10 g sample of liver was minced and homogenized in 30 mls of homogenization buffer (15 mM KH2PO4/250 mM sucrose) using a Tekmar homoginizer by pulsing 3 times 20 second pulses. This procedure was repeated for a total of 8x10 g samples of pig liver. The remaining pig liver was cut into 10-g pieces and wrapped in aluminum foil and stored at -80 °C. The homogenates from the 8 samples were pooled and centrifuged at 13,000xg for 20 minutes at 4 °C. to remove crude debris and the supernatant was further centrifuged at 100,000xg for 70 minutes at 4 °C. The microsomal pellets were resuspended into 50 mls of 150 mM KH2PO4/1 mM DTT (pH 7.4). |
| Affinity data for this assay | |
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