SDS-PAGE
Retinol-binding protein 4 (RBP4) is the specific carrier for retinol (also known as vitamin A), and is responsible for the conversion of unstable and insoluble retinol in aqueous solution into stable and soluble complex in plasma through their tight interaction. As a member of the lipocalin superfamily, RBP4 containing a β-barrel structure with a well-defined cavity is secreted from the liver, and in turn delivers retinol from the liver stores to the peripheral tissues. In plasma, the RBP4-retinol complex interacts with transthyretin (TTR), and this binding is crucial for preventing RBP4 excretion through the kidney glomeruli. RBP4 expressed from an ectopic source efficiently delivers retinol to the eyes, and its deficiency affects night vision largely. Recently, RBP4 as an adipokine, is found to be expressed in adipose tissue and correlated with obesity, insulin resistance (IR) and type 2 diabetes (T2DM). The activity of recombinant human RBP4 was measured by its ability to bind all-trans retinoic acid. The binding of retinoic acid results in the quenching of Trp fluorescence in RBP4. RBP4 was diluted to 50 ug/ml in 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5 (assay buffer) and the retinoic acid was diluted to 100, 30, 10, 3 and 1 uM in 95% ethanol. Mixing 112.5 uL of 50 ug/mL rhRBP4 and 12.5 uL of retinoic acid serial dilutions in microtubes and a blank containing 112.5 uL of 50 ug/mL rhRBP4 and 12.5 uL of 95% ethanol, then incubate at room temperature for 30 minutes. Loading 100 ul of the reaction mixtures and blank and read at excitation and emission wavelengths of 280 nm and 340 nm (top read), respectively, in endpoint mode. The result was shown, the 50% binding concentration (BC50) is >30 uM.
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