| Assay Method Information | |
| | RPE65 Retinoid Isomerase Activity Assay |
| Description: | Primary amine listed in Table 2 in DMF (1 μL) was added into a suspension containing 300 μg of RPE microsomal proteins, 1% bovine serum albumin (BSA), 2 mM disodium pyrophosphate, and 25 μM human apo-cellular retinaldehyde-binding protein (CRALBP) in 10 mM BTP buffer (200 μL) to a final concentration from 0 to 2 μM. After incubation at room temperature for 5 min, the resulting mixture was mixed with half microliter of all-trans-retinol (5 mM) in DMF, and then incubated at 37° C. for 1 h. The reaction was quenched by adding 400 μL of methanol (Fisher Chemical, Fair Lawn, NJ), and the products were extracted with 400 μL of hexanes. Production of 11-cis-retinol was quantified by normal phase HPLC using a Zorbax Rx-SIL column (5 μm, 4.6×250 mm, Agilent, Santa Clara, CA) with 10% (v/v) ethyl acetate in hexanes as the eluent at a flow rate of 1.4 mL-min−1. Retinoids were detected by monitoring their absorbance at 325 nm and quantified based on a standard curve representing the relationship between the amount of 11-cis-retinol and the area under the corresponding chromatographic peak. |
| Affinity data for this assay | |
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