Bca protein assay reagent kit

Total protein of COS-7 cells was isolated using radioimmunoprecipitation assay lysis buffer (Solarbio, Beijing, China) containing the protease inhibitor phenylmethanesulfonyl fluoride (Solarbio). The protein concentration was assessed using a bicinchoninic acid (BCA) protein assay reagent kit (Vazyme, Nanjing, China). Equal amounts of total protein (30 μg) were added to the wells of 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and separated. The proteins were transferred to polyvinylidene fluoride membranes (Millipore, Boston, USA). Subsequently, the membrane was blocked with 5% nonfat milk at room temperature for 1 h. Then, the membranes were incubated with primary antibodies against UGT1A1 and β-actin (1:1,000, Abcam, Cambridge, UK) for 12–15 h at 4°C, washed with tris-buffered saline Tween (TBST) three times, and incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G fluorescent secondary antibody (1:8,000, Abcam) at room temperature for 2 h. Fluorescence bands corresponding to UGT1A1 and β-actin were imaged using an Odyssey Fc scanner (LI-COR, NE, USA). The grayscale values of UGT1A1 and β-actin proteins were measured using LI-COR Odyssey 3.0 analytical software; the relative protein expression level is equal to the grayscale value of UGT1A1 protein/β-actin internal reference protein.

Bilirubin and its glucuronides were separated on a high-performance liquid chromatography (HPLC) column (reverse-phase Diamonsil C18 column, Dikma, Beijing, China) using a Shimadzu LC-20A HPLC system (Kyoto, Japan), and the system control and data analyses were carried out using a Shimadzu LC solution workstation (Shimadzu). The chromatographic conditions and incubation procedure for bilirubin glucuronidation were as previously described [23 (link)]. Ultrasound was used for protein extraction, and a BCA protein assay reagent kit (Vazyme) was used to assess the protein concentration. An equal amount of protein (50 μg) was added during the incubation procedure for bilirubin glucuronidation. Standard samples were prepared for calibration curves, and the final concentrations of bilirubin in the standard samples were 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, and 2 μM. The following control groups were designed: a control group containing bilirubin without uridine diphosphoglucuronic acid (UDPGA) in the reaction system and another group containing UDPGA but no bilirubin.