Extracts from HEK293FT cells expressing ubMunc13-2-EGFP or Munc13-1-EGFP, as well as non-expressing cells, were collected and lysed in RIPA buffer supplemented with Protease Inhibitor Cocktail (Invitrogen, 89900). The supernatants were collected and protein concentrations were estimated using the BCA Protein Assay Kit (Pierce 23227) after plotting the resulting BSA curve. 25 mg of protein was resolved by 4–12% SDS-PAGE (Invitrogen, Thermo Fisher Scientific) and wet-transferred onto an Amersham Hybond LFP PVDF membrane (GE Healthcare). The membrane was blotted with rabbit polyclonal α-GFP (1:300; Synaptic Systems SY132003) and HRP-conjugated mouse monoclonal α-β-Actin-Peroxidase (1:10000; Sigma-Aldrich A3854), as a loading control, followed by HRP-conjugated α-rabbit (1:2000; Agilent Dako-P0448) secondary antibody. The blot was developed by chemiluminescence Pierce ECL Plus Western blotting substrate systems (Thermofisher Scientific) and immunoreactive bands were detected using the FluorChemE image acquisition system (ProteinSimple) equipped with a cooled CCD camera.
α β actin peroxidase
Manufactured by Merck Group
α-β-Actin-Peroxidase is a lab equipment product that functions as a conjugate of actin and peroxidase. It is commonly used in various immunochemical and biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Pierce ecl plus western blotting substrate systems, by Thermo Fisher Scientific (2 mentions)
Amersham hybond lfp pvdf membrane, by GE Healthcare (2 mentions)
Hybond, by Cytiva (2 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Amersham ecl, by Cytiva (1 mentions)
3 protocols using α β actin peroxidase
1
Quantifying Ubiquitous Munc13 Isoforms
2
Western Blot Analysis of Protein Expression
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Cells were lysed in 100 mM Tris (pH 6.8), 20% glycerol, and 4% SDS buffer. Protein concentrations were determined by BCA kit (Pierce, Rockford, IL, USA), then 200 mM dithiothreitol was added boiled 5 min. Proteins were separated by SDS-PAGE and transferred to the nitrocellulose membrane. Membranes were blocked with 5% nonfat dry milk powder in 0.05% Tween20 phosphate-buffered saline (PBS-T) for 1h. Primary antibodies were incubated 2h at RT or O/N at 4°C. Membranes were washed with PBS-T and incubated 1h with secondary antibody conjugated with horseradish peroxidase. After washes blots were developed with Amersham-ECL-Plus and exposed to ChemiDoc (Bio-Rad, Hercules, CA, USA). Protein levels were evaluated by densitometric analysis using Image Lab Software (Bio-Rad). The following primary antibodies were used: α-NPM1 (ab86712; Abcam), α-βactin peroxidase (clone AC-15; Sigma A3854), and α-γ2HAx (1:400; Millipore 05-636).
3
Immunoblot Analysis of Munc13 Proteins
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Extracts from HEK293FT cells expressing ubMunc13-2-EGFP or Munc13-1-EGFP, as well as non-expressing cells, were collected and lysed in RIPA buffer supplemented with Protease Inhibitor Cocktail (Invitrogen, 89900). The supernatants were collected and protein concentrations were estimated using the BCA Protein Assay Kit (Pierce 23227) after plotting the resulting BSA curve. 25 mg of protein was resolved by 4-12% SDS-PAGE (Invitrogen, Thermo Fisher Sci-entific) and wet-transferred onto an Amersham Hybond LFP PVDF membrane (GE Healthcare). The membrane was blotted with rabbit polyclonal α-GFP (1:300; Synaptic Systems SY132003) and HRP-conjugated mouse monoclonal α-β-Actin-Peroxidase (1:10000; Sigma-Aldrich A3854), as a loading control, followed by HRP-conjugated α-rabbit (1:2000; Agilent Dako-P0448) secondary antibody. The blot was developed by chemiluminescence Pierce ECL Plus Western blotting substrate systems (Thermofisher Scientific) and immunoreactive bands were detected using the FluorChemE image acquisition system (ProteinSimple) equipped with a cooled CCD camera.
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