Western blot assays were performed as previously described.17 (link) In brief, total proteins from melanoma cells were extracted in RIPA buffer (Sigma-Aldrich; Merck KGaA) on ice (the lysis buffer contain phosphatase and protease inhibitors), Then, the lysates were prepared with lysis buffer (Beyotime), and the protein concentration was determined via the BCA assay. Approximately 50ug of protein was loaded into a 12% SDS/Polyacrylamide gel. Electrophoresis was carried out at 80 V for 20 min followed by 40 min at 120 V. Western blot analysis was performed following protein transfer to a PVDF membrane. After blocking with 5% nonfat milk, the membranes were incubated at 4 °C overnight with SOX10 (ab227684, Abcam), Notch1 (ab52627, Abcam), NICD (ab52301, Abcam), Hes1 (ab108937, Abcam), and GAPDH (ab8245, Abcam) antibody. Membranes were visualised with Electrochemiluminescence Plus Detection Reagents.
Ab227684
Manufactured by Abcam
Ab227684 is a lab equipment product. It is a device designed for use in laboratory settings. The core function of this product is to facilitate specific tasks or procedures required in a research or experimental environment.
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Lab products found in correlation
Ab108937, by Abcam (2 mentions)
Ab52627, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab52301, by Abcam (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Imagej software, by Bio-Rad (1 mentions)
2 protocols using ab227684
1
Western Blot Analysis of Melanoma Proteins
2
Protein Isolation and Western Blot Analysis
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The total protein was isolated from cells and tissues using RIPA lysis buffer (Santa Cruz, Dallas, USA). Protein samples were run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific). After blocking the PVDF membrane in TBST with 5% skim milk, add primary antibody SOX10 (ab227684, Abcam), Notch1 (ab52627, Abcam), Hes1 (ab108937, Abcam), and GAPDH (ab8245, Abcam), and incubate overnight on a shaker at 4 °C. PVDF membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. After adding luminescent solution, the protein bands were visualized using the ECL system (Thermo Fisher Scientific), and the grayscale statistics was carried out by Image J software (Bio-Rad, USA). GAPDH is used as an internal reference.
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