Bradford protein assay kit

As in previous sections, ARPE-19 cells were seeded and treated as described in Section 2.3. Then, the cells were lysed with ice-cold radio-immunoprecipitation assay (RIPA) buffer (Sigma, St. Louis, MO, USA). The total protein concentrations of the lysate supernatants were determined using the Bradford protein assay kit (Boster Biological Technology Co. Ltd., Wuhan, China). The levels of malondialdehyde (MDA), glutathione peroxidase (GSH-Px), catalase (CAT), glutathione (GSH), superoxide dismutase 2 (SOD2), and superoxide dismutase (SOD) in the supernatants were determined using their corresponding assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturers’ specifications. Data were calculated in terms of protein concentration of each sample.

Recombinant plasmids pET32a-S100A4 were transferred into E. coli BL21 (DE3) and induced to express the S100A4 protein by isopropyl-β-d-thiogalactopyranoside (IPTG). Following this, the cell lysate was sonicated and centrifuged. Protein samples from the supernatant, precipitation and cell lysate were analyzed by SDS-PAGE and stained with Coomassie brilliant blue to confirm the pET32a-S100A4 expression. The supernatant sample was purified by ion exchange chromatography column (HiTrap™ DEAE FF; loading buffer: Tris-HCl 0.02mol/l, pH 8.8; and elution buffer: 50mM/100mM/200mM/500mM NaCl Tris-HCl 0.02mol/l, pH 8.8; GE Healthcare Life Sciences, Chalfont, UK) according to the instructions of the manufacturer. Protein concentration was measured with a Bradford protein assay kit (Boster, Wuhan, China) using bovine serum albumin (BSA; Huasheng Biotech, Inc., Tianjin, China) as a standard (10 (link)).

Extracted the proteins from SW480 cells with the radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (Merk Life Sciences, Darmstadt, Germany) supplementary with phosphatase inhibitor and protease inhibitor (Sigma-Aldrich, Shanghai, China). The concentration of the extracted proteins was assessed using a commercialized Bradford protein assay kit (BOSTER Biotechnology, Wuhan, China). Protein of the same quantity (50 μg) was electrophoresly separated on 12% SDS-PAGE gel, and the protein in gels was transferred to methanol-pretreated PVDF membranes (Millipore, Darmstadt, Germany). The membranes with separated proteins on them were then submerged with 5% skim milk solution at 25℃ for 90 min, and immersed in indicated diluted primary antibodies at 4 °C for at least 12 h, followed by incubating with indicated secondary antibodies at the dilution of 1:5000 at room temperature for 90 min. In the present study, the primary antibodies against PCNA, Ki67, MMP2, and MMP9 were offered by Abcam (Cambridge, UK). Antibody against β-actin (Proteintech, Wuhan, China) was the internal control. Diluted all primary antibodies at the ratio of 1:1000. The protein bands were observed by the chemiluminescence in the enhanced ECL immunoblotting system (Tanon, Shanghai, China) and analyzed by ImageJ.