Bca protein assay kit cw0014s

The intestinal segments were weighed and placed in a 0.9% saline solution (1:9) to prepare a homogenate. The supernatant was extracted by centrifugation (2,000 × g, 20 min). The total protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (CW0014S; CoWin Biotech Co., Inc., Beijing, China). To assess antioxidant capacity, the activities of superoxide dismutase (Total-SOD) (A001-1), catalase (CAT) (A007-1), and glutathione peroxidase (GSH-Px) (A005-1), as well as total antioxidant capability (T-AOC) (A015-2) and malondialdehyde (MDA) (A003-1) levels, were quantified using colorimetric methods from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Except for MDA, which is expressed in micromoles per milligram of protein, the values are expressed in units per milligram of protein. These results were detected at specific wavelengths (SOD, 550 nm; CAT, 405 nm; GSH-Px, 412 nm; T-AOC, 520 nm; and MDA, 532 nm).

Immediately after the mice were sacrificed, the uterus was rapidly dissected, and the embryos were removed, frozen in liquid nitrogen, and stored. The uterus with removed embryos was weighed and placed in a 0.9% saline solution (1:9) to prepare a uterine homogenate. Finally, supernatants were extracted by centrifugation (2000× g, 20 min). The total protein concentration was measured using a BCA Protein Assay Kit (CW0014S, CoWin Biotech Co., Inc., Beijing, China). Total antioxidant capacity (T-AOC) (A015), SOD (A001-1), GSH-Px (A005), and MDA (A003-1) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). GSH-Px and SOD are well-known scavenger enzymes that protect cells from oxidative stress. SOD was detected by the xanthine oxidase method, and GSH-Px was determined by the rate at which it was converted to the enzymatic reaction of oxidized glutathione disulfide (GSSG). T-AOC was detected by converting Fe³⁺ to Fe²⁺. Those values were expressed as U/mg of protein. MDA is responsible for inducing oxidative stress, and it reacts with thiobarbituric acid to form a red complex and is expressed as nmol/mg of protein. These results were detected at specific wavelengths (T-SOD: 550 nm, GSH-Px: 412 nm, T-AOC: 520 nm, and MDA: 532 nm). Five tissue samples were included in each group, and each sample was tested in triplicate.

Proteins extracted from the colon tissue were separated by 8–12% SDS-PAGE after the concentration was detected by the BCA protein assay kit (CW0014S, CoWin Biotech Co., Inc., Beijing, China). They were then transferred to 0.2 μm polyvinylidene fluoride membranes (Merck KGaA Co., Ltd., Darmstadt, Germany). Membranes were blocked with 5% skim milk for 1.5 h. After blocking, the membranes were incubated with different primary antibodies at 4 ℃ overnight. Claudin 1 (1/3000, Abcam Co., Inc., Cambridge, UK), Claudin 3 (1/1000, Abcam Co., Inc., Cambridge, UK), Occludin (1/1000, Abcam Co., Inc., Cambridge, UK), ZO-1 (1/1000, Abcam Co., Inc., Cambridge, UK), TRL-4(1/1000, Proteintech Co., Wuhan, China), IKB(1/1000, Abmart Co., Inc, Shanghai, China), p65(1/5000, Abmart Co., Inc, Shanghai, China), p-IKB(1/1000, Abcam Co., Inc., Cambridge, UK), pp65(1/1000Abcam Co., Inc., Cambridge, UK), β-actin(1/1000, LABLEAD Biotech Co., Ltd., Beijing, China). Then, the membranes were washed with Trisbuffered saline Tween (TBST) and then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG, or goat anti-rabbit IgG (CoWin Biotech Co., Inc., Beijing, China) for 1.5 h. The membranes were imaged with a Tanon 5200 imaging system. (Tanon Science & Technology Co., Ltd., Shanghai, China)