After HDACis treatment, cells were harvested, washed twice with PBS and lysed using RIPA lysis buffer (25 mM TrisHCL pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodiumdeoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail (Roche). Protein extract samples were quantified using the Bradford method (Bio-Rad Protein Assay Dye Reagent Concentrate, Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Total protein extract samples were separated by 15% SDS-PAGE and transferred to nitrocellulose membranes. After blocking, proteins were probed with the following primary antibodies: anti-acetylhistone H3 (Lys9, Lys14) antibody (polyclonal, rabbit, 1:2500 dilution, Thermo Fisher Scientific, Rockford, IL, USA), anti-histone H3 (polyclonal, rabbit, 1:1000 dilution, Thermo Fisher Scientific), and then with Peroxidase-AffiniPure Anti-Rabbit IgG antibody (polyclonal, goat, 1:10000 dilution, Jackson ImmunoResearch, PA, USA) as a secondary antibody. Proteins were detected by chemiluminescence using Luminata Forte Western HRP (Merck Millipore, Darmstadt, Germany) and acquired using the ChemiDoc XRS+ imaging system (Bio-Rad).
Luminata forte western hrp
Manufactured by Merck Group
Sourced in Germany
Luminata Forte Western HRP is a laboratory equipment product for performing Western blot analysis. It is designed to detect and quantify specific proteins in complex biological samples using a chemiluminescent detection method.
Automatically generated - may contain errors
Lab products found in correlation
Hybond c extra, by GE Healthcare (2 mentions)
Hybond, by Cytiva (2 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Protein assay dye reagent concentrate, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Anti histone h3, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit hrp conjugate, by Bio-Rad (1 mentions)
3 protocols using luminata forte western hrp
1
Detecting Histone H3 Acetylation
2
Western Blot Analysis of E. coli and Y. pseudotuberculosis
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Cell pellets of E. coli DH5α, Y. pseudotuberculosis YPIII, Y. pseudotuberculosis YP216 were resuspended in 1 x SDS sample buffer (2% (w/v) SDS, 0.1% (w/v) bromophenol blue, 10% glycerol, 50 mM Tris/HCl, pH 6.8) according to their optical density (CNFY synthesis studies: 100 μl for OD600 = 1; gfp reporter gene assay: 100 μl for OD600 = 0.5). After boiling for 10 min at 95°C, samples were centrifugated (10 min, 13000 rpm) and the protein extracts were subjected to SDS gel electrophoresis (10–12.5% SDS PAA gels). Subsequent Western transfer was performed by tank blotting onto a nitrocellulose membrane (Hybond-C Extra, GE Healthcare, Munich, Germany). An anti-CNFY antibody was used in a 1:1000 dilution, anti-GFP antibody (ABIN129570, antibodies-online GmbH, Aachen) was used in a 1:10000 dilution and secondary antibody goat anti-rabbit-HRP conjugate (Bio-Rad, Munich, Germany) in a 1:3000 dilution. Luminescence signals were detected by incubating membranes with Luminata Forte Western HRP (Merck, Darmstadt, Germany) substrate and the ChemiImager Ready (Alpha Innotec, San Leandro, USA).
3
GFP Expression Analysis in E. coli
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E. coli cells harboring the pBAD2-gfp plasmids were grown overnight in 5 ml M9 minimal medium at 25, 30 or 37°C. Fifteen milliliters M9 minimal medium with ampicillin andl -arabinose were pre-warmed to 25, 30 or 37°C and inoculated with the overnight culture to an optical density (OD600) of 0.05. After growth to an optical density (OD600) of 0.5 1 ml of the culture was harvested (1 min, 13 000 rpm). Pellets were resuspended in TE buffer (10 mM Tris, pH 8, 1 mM EDTA; 100 μl TE buffer per optical density OD600 of 0.5) and mixed with protein sample buffer (final concentrations of 2% SDS (w/v), 0.1% (w/v) bromophenol blue, 10% glycerol (v/v), 1% β-mercaptoethanol, 50 mM Tris–HCl, pH 6.8). After incubation for 5 min at 95°C, samples were centrifuged and separated via SDS-PAGE. Western transfer was performed by tank blotting onto a nitrocellulose membrane (Hybond™-C Extra, GE Healthcare, Munich, Germany). GFP antibody (ABIN129570, antibodies-online GmbH, Aachen) was used in an 1:10 000 dilution, secondary antibody goat anti rabbit-HRP conjugate (Bio-Rad, Munich, Germany) in 1:3000 dilution. Luminescence signals were detected using Luminata Forte Western HRP (Merck, Darmstadt, Germany) substrate and the ChemiImager Ready (Alpha Innotec, Kasendorf, Germany).
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E. coli cells harboring the pBAD2-gfp plasmids were grown overnight in 5 ml M9 minimal medium at 25, 30 or 37°C. Fifteen milliliters M9 minimal medium with ampicillin and
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