For Western blot analysis, 5 × 106 cells were harvested and proteins were solubilized as described.48 (link) 30 µg protein/lane was separated in 10% SDS-PAGE gels, transferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) and stained with Ponceau S as previously described.45 (link) Membranes were incubated overnight at 4°C with a primary polyclonal antibodies directed against BGN (Proteintech, Rosemont, IL, USA) and K-RAS (Cell Signaling Technology, Danvers, USA) and monoclonal antibodies directed against β-actin (Sigma-Aldrich, St. Louis, USA) and/or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology), respectively, followed by incubation for 1 h with a horseradish peroxidase (HRP) linked secondary antibody (Ab, Cell Signaling Technology) and developed using the ECL method. Chemiluminescence signals were visualized with the Lumi-Light Western Blotting Substrate (Roche Diagnostics) and recorded with a LAS3000 system (Fuji, Tokyo, Japan). To quantify the protein expression, the appropriate area of the signal was integrated using an AIDA image analyzer (Raytest, Straubenhardt, Germany) and subsequently normalized to β-actin or GAPDH.
β actin
Manufactured by Elysia raytest
Sourced in Germany
β-actin is a highly conserved cytoskeletal protein that is ubiquitously expressed in eukaryotic cells. It is a major component of the actin cytoskeleton and plays a crucial role in various cellular processes, such as cell motility, structure, and signaling.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (3 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Gapdh, by Elysia raytest (2 mentions)
Lumi light western blotting substrate, by Roche (2 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (1 mentions)
Las 3000 system, by Fujifilm (1 mentions)
Aida image analyzer, by Elysia raytest (1 mentions)
Hif 2α, by Novus Biologicals (1 mentions)
Las3000, by Elysia raytest (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Gapdh, by Merck Group (1 mentions)
Las 3000 ccd camera system, by Fujifilm (1 mentions)
Aida software, by Elysia raytest (1 mentions)
3 protocols using β actin
1
Western Blot Protein Quantification Protocol
2
Western Blot Analysis of Protein Expression
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Cells were harvested as previously described [13 (link)] and protein isolated according to Laemmli [15 (link)]. Fifty μg protein/lane were subjected to Western blot analysis as recently described [13 (link)]. Membranes were incubated over night at 4°C with the primary monoclonal antibodies (mAb) directed against succinate dehydrogenase complex subunit A (SDHA; Cell Signaling), HIF2α (Novus) and β-actin (Sigma-Aldrich), followed by incubation for 1 h with horseradish peroxidase-linked secondary antibody and developed using the ECL method. Chemoluminescence signals were detected using a CCD camera (LAS3000, Raytest). For quantification of the SDHA protein expression the respective area of the signal was integrated using an AIDA image analyser (Raytest) and subsequently normalized to β-actin.
3
Western Blot Analysis of Protein Expression
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Aliquots of 50 μg of solubilized protein/lane were separated on 12% SDS-PAGE gels and subsequently transferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Membranes were processed as previously described [54 (link)] using target protein-specific primary antibodies directed against beta-2-microglobulin (B2M) (kindly provided by Dr. Soldano Ferrone; [55 (link)]), MnSOD2 (Abfrontier, Aachen, Germany), GAPDH (Cell signaling) or β-actin (Sigma-Aldrich) in combination with horse-radish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling, Frankfurt, Germany). Protein bands were visualized with LumiLight Western Blotting substrate (Roche, Mannheim, Germany) and recorded with a LAS 3000 CCD camera system (FUJIFILM, Duesseldorf, Germany). The co-detection of the GAPDH or β-actin signal, respectively, for each lane on the given blot served as loading control and the relative protein expression level for each target were defined using AIDA software (Raytest, Sprockhoevel, Germany).
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