Ecl western blotting detection kit goat igg

According to the previous studies [19 ,20 ], total protein samples were harvested from brain tissues by a Total Protein Extraction Kit (BC3711, Solarbio), and measured with the BCA Protein Assay Kit (PC0020, Solarbio). Then, protein samples were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene fluoride (PVDF) membranes (IPVH00010, EMD Millipore, Billerica, MA, USA). The membranes were sealed with western blocking buffer (SW3010, Solarbio) and hatched with primary antibodies at 4 °C overnight. Bounds were determined with goat anti-rabbit IgG H&L (HRP) (ab6721, 1:10000, Abcam) and ECL Western Blotting Detection Kit (Goat IgG) (SW2030, Solarbio). The band intensity was examined via QUANTITY ONE software (Bio‐Rad, Hercules, CA, USA). The expression level of each protein was shown after being normalized with GAPDH. The primary antibodies contained anti-PIAS1 (1:10000, ab109388, Abcam, Cambridge, UK), anti-Bax (1:5000, ab32503, Abcam), anti-Bcl-2 (1:2000, ab196495, Abcam), anti-cleaved caspase3 (1:1000, 9661, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-p65 (1:1000, 4764, Cell Signaling Technology), anti-phosphorylated p65 (p-p65) (1:1000, 3031, CST), anti-IκBα (1:1000, 9242, CST), anti-p-IκBα (1:1000, 2859, CST) and anti-GAPDH (1:10000, ab181602, Abcam).

Western blot assays were performed as the previous methods.^{28 (link),29 (link)} Total proteins were prepared from Huh7 and HCCLM3 cells, as well as tumors samples through the introduction with RIPA lysis buffer (P0013B, Beyotime) and quantified with BCA kit (P0012S, Beyotime) in accordance with the operation instruction. 20 μg protein samples were dissolved with 10% SDS-PAGE, and electrically shift onto PVDF membranes for the conventional operations of western blot experiment. The primary and second antibodies included anti-BTF3 (1:1000, ab203517, Abcam, Cambridge, UK), anti-FOXM1 (1:2000, ab180710, Abcam), anti-GLUT1 (1:1000, ab128033, Abcam), anti-β-actin (1:1000, ab8227, Abcam) and goat anti-rabbit IgG H&L (HRP) (ab6721, 1:10000, Abcam). The membranes were developed with ECL Western Blotting Detection Kit (Goat IgG) (SW2030, Solarbio), and the band intensity was determined by ImageJ software (National Institutes of Health, USA).