Cell lysates were prepared using ReadyPrep Protein Extraction Kit (Bio-Rad, Hercules, CA) according to the instructions provided. Total cell lysates (50μg) were separated electrophoretically by a 10% polyacrylamide SDS-PAGE gel and transferred onto a polyvinylidene fluoride membrane using the BioRad Mini Protean transfer system. The blots were then blocked with 5% skim milk in PBST for 1 h and probed with primary antibodies overnight at 4°C. All primary antibodies were purchased from Cell Signaling unless otherwise specified. The membranes were sequentially detected with an appropriate peroxidase-conjugated secondary antibody incubated at room temperature for 1 h. Blots were washed 3 times with PBS. Signals were then detected using an enhanced chemiluminescence detection system and BioSpectrum Imaging System (UVP, Upland, CA).
Mini protean transfer system
Manufactured by Bio-Rad
Sourced in Germany
The Mini Protean transfer system is a compact and efficient electrophoresis unit designed for the transfer of proteins from polyacrylamide gels to membranes. It features a simple and user-friendly design to facilitate the blotting process.
Automatically generated - may contain errors
Lab products found in correlation
Readyprep protein extraction kit, by Bio-Rad (2 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Biospectrum imaging system, by Analytik Jena (1 mentions)
Ab77735, by Abcam (1 mentions)
Ab155091, by Abcam (1 mentions)
Ab183727, by Abcam (1 mentions)
Ab90867, by Abcam (1 mentions)
Ab7291, by Abcam (1 mentions)
Ab1791, by Abcam (1 mentions)
4 protocols using mini protean transfer system
1
Western Blot Analysis of Protein Lysates
2
Protein Extraction and Quantification Protocol
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Total protein was extracted from samples collected in both 2010 and 2011 growing seasons by the previously described protocol of Wang et al. (2006) (link) with slight modifications. Samples (0.5 g) were first homogenized in liquid nitrogen in the presence of polyvinylpolypyrrolidone (PVPP). The protein pellets obtained were dissolved in SDS-PAGE sample buffer containing 0.15 M Tris (pH 6.8), 1.2% SDS, 30% glycerol, 2.14 M β-mercaptoethanol (Sigma-Aldrich, St Louis, MO, United States). Extracted proteins were quantified by band intensities confirmed by fractionating on 10% SDS-PAGE gel, and staining with Coomassie protein staining buffer (0.1% Coomassie Brilliant Blue R-250, 50% methanol and 10% glacial acetic acid). Equal amounts of proteins were separated by 10% SDS-PAGE and transferred to PROTEAN nitrocellulose transfer membrane (Whatman GmbH, Dassel, Germany) using the Mini-Protean Transfer system (Bio-Rad Laboratories, Hercules, CA, United States). Immunoblot assays are as previously described (Acheampong et al., 2015 (link)). Band intensities were analyzed using ImageJ 1.48v software (Schneider et al., 2012 (link)).
3
Western Blot Analysis of CD133, MGMT, and Tubulin
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After treatment, the cell lysates were prepared using ReadyPrep Protein Extraction Kit (Bio-Rad) according to instructions provided. Total cell lysates (20 μg) were separated electrophoretically by a 10% polyacrylamide SDS-PAGE gel and transferred onto a polyvinylidene fluoride membrane using the Bio-Rad Mini-Protean transfer system. The membrane was cropped and further incubated overnight at 4°C with specific antibodies against CD133 (St John's Laboratory Ltd, London, UK), MGMT (Cell Signaling Technology, Beverly, MA, USA) and tubulin (Abcam, Cambridge, MA, USA), respectively. After incubation with primary antibodies, the cropped membranes were washed with TBST 3 times and then incubated with horseradish peroxidase-labelled secondary antibody for 90 minutes at room temperature. After washing with TBST 3 times, final detection was performed with enhanced chemiluminescence (ECL) Western blotting reagents (Minipore) and BioSpectrum Imaging System (UVP, Upland, CA, USA).
4
Proteasome Subunit Quantification by Western Blot
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