Protein lysates were prepared in the RIPA buffer (radio immunoprecipitation assay lysis buffer) supplemented with protease inhibitor cocktail and phosphatase inhibitors (Thermo Scientific, Waltham, MA). The total protein concentration of the soluble extract was determined using the BCA protein assay Kit (Thermo Scientific). Each protein sample (30ug) was resolved to SDS–PAGE, transferred onto a polyvinylidenedifluoride membrane (Millipore) and incubated overnight at 4°C with primary antibodies. The antibodies used were: ERG (1:2000 Abcam, #ab133264); JUN (1:1000 Santa Cruz, sc-1694X); rabbit anti-FLAG (1:1000 Sigma, F7425); pan-cytokeratin (1:1,000 BioLegend, clone C-11 cat # 628602 and Lot # B181240);α-tubulin (1:1,000 Sigma, #T6199) and GAPDH (1:10,000 Abcam, sb-75834). Following three washes with TBS-T, the blot was incubated with horseradish peroxidase-conjugated secondary antibody and immune complexes were visualized by enhanced chemiluminescence detection (ECL plus kit, GE Healthcare, UK). The blot was reprobed with monoclonal antibody against beta-actin (1:10,000 Sigma). Total protein was extracted and separated by gel electrophoresis. Protein was then transferred to nitrocellulose membranes and probed overnight using the appropriate primary antibodies.
Monoclonal antibody against beta actin
Manufactured by Merck Group
Sourced in United States
Monoclonal antibody against beta-actin is a laboratory reagent used for the detection and quantification of the beta-actin protein, which is a common and widely expressed cytoskeletal protein. This antibody can be used in various techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to visualize and analyze the presence and distribution of beta-actin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Ecl plus kit, by GE Healthcare (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab133264, by Santa Cruz Biotechnology (2 mentions)
Sc 1694x, by Merck Group (2 mentions)
F7425, by BioLegend (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Animal free blocker, by Vector Laboratories (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Primary antibody against s100a9, by Abcam (1 mentions)
Anti rat hrp conjugated secondary antibody, by Merck Group (1 mentions)
3 protocols using monoclonal antibody against beta actin
1
Western Blot Analysis of Protein Expression
2
Protein Quantification in Murine Skin
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Samples of murine skin were lysed in lysis buffer containing 1% Triton X-100, 1 M Tris-HCl (pH 7.4), 0.5 M EDTA, 2 M NaCl, 10% Na-deoxycholate, 10% sodium dodecyl sulfate (SDS), and glycerol for 30 minutes at room temperature. Proteins were separated by size on 15% polyacrylamide SDS gels (PAGE) and transferred to nitrocellulose membranes (BioRad, CA, USA) by electroblotting. After the membranes were blocked with 1x Animal-Free Blocker (Vector Laboratories, Inc., Burlingame, CA, USA) for 1 hour, they were incubated overnight with a primary antibody against S100A9 (1 : 5000) (Abcam, Cambridge, UK) and subsequently incubated with an anti-rat HRP-conjugated secondary antibody (1 : 15000) (Sigma-Aldrich, MO, USA). Signals were developed and visualized using the Clarity Western ECL Substrate (BioRad, CA, USA). The intensity of the protein band was analyzed using the ImageJ software program (version 1.41). Equal loading and transfer of the proteins were assessed using a monoclonal antibody against beta-actin (1 : 1000) (Sigma-Aldrich, MO, USA).
3
Western Blot Protein Expression Analysis
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Protein lysates were prepared in the RIPA buffer (radioimmunoprecipitation assay lysis buffer) supplemented with protease inhibitor cocktail and phosphatase inhibitors (Thermo Scientific, Waltham, MA). The total protein concentration of the soluble extract was determined using the BCA protein assay Kit (Thermo Scientific). Each protein sample (30μg) was resolved to SDS–polyacrylamide gel electrophoresis (SDS–PAGE), transferred onto a polyvinylidene difluoride membrane (Millipore) and incubated overnight at 4 °C with primary antibodies. The antibodies used were ERG (1:2,000 Abcam, #ab133264); JUN (1:1,000 Santa Cruz, sc-1694X); rabbit anti-FLAG (1:1,000 Sigma, F7425); pan-cytokeratin (1:1,000 BioLegend, clone C-11 cat # 628602 and Lot # B181240); αtubulin (1:1,000 Sigma, #T6199) and GAPDH (1:10,000 Abcam, sb-75834). Following three washes with TBS-T, the blot was incubated with a horseradish peroxidase-conjugated secondary antibody and immune complexes were visualized by enhanced chemiluminescence detection (ECL plus kit, GE Healthcare, UK). The blot was reprobed with a monoclonal antibody against beta-actin (1:10,000 Sigma). Total protein was extracted and separated by gel electrophoresis. Protein was then transferred to nitrocellulose membranes and probed overnight using the appropriate primary antibodies.
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