Bca protein concentration determination kit g2026

The liver tissue of each mouse was ground into homogenate and centrifuged to obtain the supernatant. The total protein concentration of the supernatant was determined using a BCA protein concentration determination kit G2026 (Servicebio, Wuhan, China) according to the manufacturer’s instructions. The TG content in liver samples was then measured using the TG Determination Kit (GPO-PAP method) (C061, Huili) against the total protein concentration [18 (link)]. In detail, a 250 μL GPO-PAP working solution was mixed with 2.5 μL PBS, standard glycerol (1.7 mmol/L), or supernatant. The mixture was subjected to OD analysis at 510 nm using the automatic biochemistry analyzer. The TG concentration was resultantly determined using the colorimetric method with standard glycerol normalized by PBS.

Protein samples were harvested from mouse liver using RIPA lysis buffer and quantified by BCA protein concentration determination kit G2026 (Servicebio) with the same method. Total protein extracts were incubated with loading buffer at 100°C for 10 min and subjected to electrophoresis on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently, proteins were transferred onto polyvinylidene difluoride membranes. The membranes were incubated with protein-free rapid blocking buffer for 20 min, and subsequently, with primary antibodies against a-tubulin (1:1,000, 66031, Proteintech, Rosemont, IL, United States), AMPK-α1 (1.5:1,000, AF6422, Affinity, Liyang, China), SREBP1 (1.5:1,000, AF6283, Affinity), and FASN (1:1,000, 10624, Proteintech) overnight at 4 °C, and with horseradish peroxidase (HRP) -conjugated secondary antibodies at room temperature for 1 h. Protein bands were visualized by chemiluminescence using AI680 (Cytiva, Marlborough, MA, United States) and assessed using ImageJ software (https://imagej.nih.gov/ij/) with integrated density/area.