Total proteins were prepared by resuspending cells in RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.1% sodium dodecyl sulphate (SDS), 0.5% sodium deoxycholate, 1% NP-40, 1 mM EDTA, protease and phosphatase inhibitors [Roche]). Protein concentration was determined using a BCA assay (ThermoFisher Scientific). The cell lysate was denatured at 95°C for 5 min. The cell lysates were resolved on 4–15% TGX gradient gels (Bio-Rad Laboratories) and transferred to nitrocellulose membrane (Amersham). Membranes were blocked with 5% non-fat dried milk in TBS with 0.2% Tween for 1 hr at RT and then incubated with primary antibody overnight at 4°C. Primary antibodies used were against MF20 (MyHC) (DSHB, Mf20-s; RRID:AB_2147781 ), G9a (Abcam, ab185050; RRID:AB_2792982 ), Ezh2 (Cell Signaling, #3147; RRID:AB_10694383 ), Wnt7b (Abcam, ab94915; RRID:AB_10675749 ), Cyclin D3 (Santa Cruz biotechnology, sc-182; RRID:AB_2259653 ), and GAPDH (Sigma, G9545; RRID:AB_796208 ). After washing in TBS with 0.2% Tween, membranes were incubated in HRP-conjugated specific secondary antibody (goat anti-rabbit-anti-mouse [IgG-HRP Santa Cruz Biotechnologies]) for 1 hr at RT. After washing in TBS with 0.2% Tween, blots were developed with Western lightning enhanced chemiluminescence (ThermoFisher Scientific), the signal detection was performed with the use of ChemiDoc (Bio-Rad).
Western lightning enhanced chemiluminescence
Manufactured by Thermo Fisher Scientific
Sourced in United States
Western Lightning Enhanced Chemiluminescence is a lab equipment product designed to detect and quantify proteins in Western blot analysis. It provides a sensitive and reliable method for chemiluminescent detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bca assay, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Tgx gradient gel, by Bio-Rad (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Ab94915, by Santa Cruz Biotechnology (1 mentions)
G9545, by Merck Group (1 mentions)
Radioimmunoprecipitation assay buffer, by Cell Signaling Technology (1 mentions)
2 protocols using western lightning enhanced chemiluminescence
1
Protein Extraction and Western Blot Analysis
2
Quantifying Intracellular Protein Levels
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Radioimmunoprecipitation assay buffer was used for the intracellular protein extraction (Cell Signalling Technology, Danvers, MA, USA). Protein (50 µg) contents were obtained by employing the sodium dodecyl-sulfate polyacrylamide gel electrophoresis technique. Next, the obtained protein content was loaded onto polyvinylidene difluoride membranes and subjected to the appropriate primary and secondary antibody treatments. The increased chemiluminescence approach was used in the process of developing the membranes (Pierce, Rockford, IL, USA). After being subjected to X-ray film, the bands were observed using Western Lightning Enhanced Chemiluminescence (Thermo Fisher Scientific, MA, USA). Each experiment was repeated 3 times to validate the obtained results. The Phoretix 1D program was then used to analyze the results.
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