Lc solution workstation

Gel permeation chromatography (GPC) was used to determine the MW distribution of the SPI samples. SPI solution (2%, w/v) was centrifuged at 10,000g for 15 min. The supernatant was diluted to 10 mg/ml with distilled water and filtered through a 0.45‐μm filter. Protein separation was performed with 10 μL of the diluted SPI supernatant in an HPLC instrument (LC‐20A; Shimadzu Co., Tokyo, Japan) equipped with a PROTEIN KW‐804 column (8.0 × 300 mm; Shodex, Tokyo, Japan) and a UV‐detector (SPD‐M20A, Shimadzu Co., Tokyo, Japan) (280 nm). The column has an exclusion size limit of 1,000 kDa for globulins. Phosphate buffer (50 mM, pH 7.0) containing 0.3 M NaCl was used as eluent; the flow rate was set to 1.0 ml/min and the eluate was monitored at 280 nm. All samples were measured in duplicate; representative results were selected for analysis and discussion. An LCsolution Workstation (Shimadzu Co., Tokyo, Japan) was used to analyze the area under the peaks. To estimate the MW of protein particles and aggregates in SPI samples, a standard curve was constructed using the following MW markers: thyroglobulin (660 kDa), amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome C (12 kDa).

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.