Cells were lysed at 48 h post-transfection with BROD (Thermo Fisher Scientific) buffer for 30 min on ice, then denatured with loading buffer boiled at 100°C for 10 min. Protein samples were separated by SDS-PAGE gel electrophoresis and transferred onto a PVDF membrane. Membranes were blocked with 5% skim milk at 37°C for 1 h, then incubated with primary antibodies at 4°C overnight. Primary antibodies included KDM5B (A7772, 1:1000, ABclonal, Wuhan, Hubei, China) and GAPDH (CW0100M, 1:5000, CWBIO, Nanjing, Jiangsu, China). After rinsing three times in TBST, the membranes were incubated at 37°C for 1 h with HRP-conjugated secondary antibodies (BE0101-100 or BE0102-100 Easybio, Beijing, China). After rinsing three times in TBST, membranes were treated with super ECL Plus Western Blotting Substrate (Thermo Fisher Scientific) and signals were visualized using a Tanon 5200 (Tanon Science & Technology Co. Ltd, Shanghai, China).
Super ecl plus western blotting substrate
Manufactured by Thermo Fisher Scientific
Sourced in China
Super ECL Plus Western Blotting Substrate is a chemiluminescent detection reagent used for quantifying proteins in western blot analysis. It generates a stable luminescent signal that can be detected using a compatible imaging system.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Be0101 100, by EASYBIO (1 mentions)
Anti sqstm1, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Anti xpo1, by Cell Signaling Technology (1 mentions)
Anti p chek1, by Cell Signaling Technology (1 mentions)
Anti p atm, by Cell Signaling Technology (1 mentions)
Anti rnf168, by Abcam (1 mentions)
Anti rad51, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Anti γh2ax, by Cell Signaling Technology (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
2 protocols using super ecl plus western blotting substrate
1
Western Blot Analysis of KDM5B and GAPDH
2
Molecular Profiling of GBM Cells
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GBM cells were lysed with RIPA lysis buffer (Thermo Fisher Scientific, 89900) containing 1% protease inhibitors (Thermo Fisher Scientific, A32953). Proteins concentration was measured using BCA protein assay (Thermo Fisher Scientific, 23225) reagents. A total of 20 μg of proteins were separated by 10% or 12% SDS-PAGE and transferred to polyvinylidene fluoride/PVDF membranes (Bio-Rad, 162-0177). Then, after blocked with 5% skim milk for 60 min, membranes were incubated at 4°C overnight with primary antibodies as follow: anti-XPO1 (Cell Signaling Technology, 46249S), anti-p-ATM (Cell Signaling Technology, 5883S), anti-p-CHEK1 (Cell Signaling Technology, 2348S), anti-p-CHEK2 (Cell Signaling Technology, 2661), anti-RAD51 (Cell Signaling Technology, 8875), anti-γH2AX (Cell Signaling Technology, 9718S), anti-SQSTM1 (Cell Signaling Technology, 88588), anti-LC3 (MBL BEIJING BIOTECH CO., LTD, M186-3), anti-RNF168 (Abcam, ab271099). Next the membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, 7074s). The signal was visualized using Super ECL Plus western blotting Substrate (Thermo Fisher Scientific, 34577) and detected by a ChemiDocXRS system (Bio-Rad).
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