Total proteins were extracted from cells using the CHAPS lysis buffer (Beyotime, China). The quantification of protein was measured by the Bradford assay kit (Sigma). Equal amount of protein (10 μg) was loaded onto 10% SDS-PAGE and transferred to PVDF membrane (Millipore). The membrane was blocked with 5% skimmed milk for 1 h at room temperature. Then, the membrane was incubated with primary antibody at 4°C overnight. The following antibodies were used: phospho-ERK (cat: 4370; dilution: 1 : 1000; Cell Signaling Technology), ERK (cat: 4696; dilution: 1 : 1000; Cell Signaling Technology), phospho-p38 (cat: 4511; dilution: 1 : 1000; Cell Signaling Technology), p38 (cat: 8690; dilution: 1 : 1000; Cell Signaling Technology), phospho-FRS2 (cat: 3864; dilution: 1 : 1000; Cell Signaling Technology), FRS2 (cat: ab183492; dilution: 1 : 1000; Abcam), NOD2 (cat: ab31488; dilution: 1 : 1000; Abcam), EZH2 (cat: ab191250; dilution: 1 : 1000; Abcam), H3K27me3 (cat: ab6002; dilution: 1 : 1000; Abcam), and GAPDH (cat: ab8245; dilution: 1 : 5000; Abcam). Secondary antibodies conjugated to horseradish peroxidase were ordered from Sigma-Aldrich (USA). The results were visualized using Tanon™ High-sig ECL Western Blotting Substrate (Tanon, China) in Tanon-4600 instrument (Tanon, China). All experiments were repeated at least three times.
Chaps lysis buffer
Manufactured by Beyotime
Sourced in China
CHAPS lysis buffer is a detergent-based solution designed for cell lysis and protein extraction. It facilitates the disruption of cell membranes to release intracellular proteins. The buffer contains the zwitterionic detergent CHAPS, which aids in solubilizing a wide range of proteins while maintaining their native structure and function.
Automatically generated - may contain errors
Lab products found in correlation
Secondary antibodies conjugated to horseradish peroxidase, by Merck Group (1 mentions)
Phospho frs2, by Cell Signaling Technology (1 mentions)
H3k27me3, by Abcam (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
Bradford assay kit, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Image pro plus software version 6, by Media Cybernetics (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
2 protocols using chaps lysis buffer
1
Protein Expression Analysis by Western Blot
2
Vernodalol-Induced Apoptosis Pathway Analysis
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The cells were treated with vernodalol at different concentrations (0, 50, 75 or 100 µM) for the indicated time (24 h). The cells were extracted and lysed with CHAPS lysis buffer (Beyotime Institute of Biotechnology, Beijing, China) for 30 min on ice and then centrifuged at 12,000 × g for 15 min at 4°C. The total protein concentration was determined with bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Equal amounts (30 µg per lane) of protein samples were subjected to 8–12% SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Each membrane was blocked with 10% non-fat milk at room temperature for 2 h and incubated with primary antibodies (caspase-9, caspase-3, PARP, Bcl-2, Bax, Bcl-2, Bad, Bak, PTEN, Bim, cytochrome c, Smac/DIABLO, Akt, PI3K, mTOR and GAPDH) at 4°C overnight, followed by incubation with goat anti-mouse (cat. no. SZ200) or anti-rabbit (cat. no. SZ2004) secondary antibodies (1:1,000 dilution; Santa Cruz Biotechnology, Inc.) conjugated to horseradish peroxidase at room temperature for 2 h. The relative protein expression levels were quantified using Image-Pro Plus software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) and normalized to GAPDH.
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