Nuclear extracts or cell lysates (25 μg/lane) were loaded in 1x NuPage loading buffer (Thermo Fisher Scientific, cat # NP0007) onto 12% NuPage® Bis-Tris gels (Thermo Fisher Scientific, cat # NP0336BOX) alongside a Precision Plus Protein dual colour standard marker (Bio-Rad, cat # 1610374). Following standard electrophoresis using 1x MOPS running buffer and transfer, nytran membranes were incubated for 1 hr in blocking solution (TBST, 5% BSA) and probed overnight at 4 °C with mouse monoclonal anti-Histone H3 (abcam, cat # ab10799, 1 in 1000 dilution) or rabbit polyclonal anti-H3K27me3 (Thermo Fisher Scientific, cat # PA5-31817, 1 in 1000 dilution). Detection of EZH2 was performed using rabbit monoclonal anti-EZH2 (Cell Signaling Technology, cat # 5246, 1 in 500 dilution) with normalization against the endogenous control GAPDH (Santa Cruz, cat # sc-47724, 1 in 1000 dilution). Following extensive washes in 1x TBST, membranes were incubated in goat anti-mouse or rabbit IgG –alkaline phosphatase (Sigma Aldrich, 1 in 10000 in blocking solution). Membranes were washed 3 times for 15 min in TBST, followed by signal development in BCIP/NBT liquid substrate (Sigma Aldrich, cat # B1911) with image recording using an Alpha Innotech gel imager (Fluor Chem ® HD2, BioZym), n = 3.
Goat anti mouse or rabbit igg alkaline phosphatase
Manufactured by Merck Group
Goat anti-mouse or rabbit IgG–alkaline phosphatase is a secondary antibody conjugate used in various immunoassay techniques. It is a goat-derived antibody that recognizes and binds to mouse or rabbit primary antibodies, and is conjugated with the enzyme alkaline phosphatase. This conjugate can be used to detect and quantify target antigens in samples by catalyzing a colorimetric or luminescent reaction.
Automatically generated - may contain errors
Lab products found in correlation
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Anti h3k27me3, by Thermo Fisher Scientific (1 mentions)
Bcip nbt liquid substrate, by Merck Group (1 mentions)
Mouse monoclonal anti histone h3, by Abcam (1 mentions)
Nupage loading buffer, by Thermo Fisher Scientific (1 mentions)
Anti ezh2, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
2 protocols using goat anti mouse or rabbit igg alkaline phosphatase
1
Histone H3 and H3K27me3 Western Blot
2
Protein Extraction and Immunoblotting
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Cells were harvested at indicated time points by centrifugation at 4000× g for 2 min. Subsequently, cell pellets were resuspended in Milli-Q H 2 0 and boiled for at least 5 min in 5 × SDS loading buffer (50 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 0.02% (w/v) bromophenol blue, 5% (v/v) β-mercaptoethanol). Proteins were visualised either by Coomassie Brilliant Blue staining or by immunoblotting against the protein of interest using standard methods [21] . PVDF membranes were probed with either anti GFP rabbit polyclonal (Abcam), anti β-galactosidase rabbit polyclonal (Life Technologies, Carlsbad, CA, USA), anti-Histidine-tagged mouse monoclonal (Bio-Rad AbD Serotec, Raleigh, NC, USA), or anti Strep-tag classic mouse monoclonal (Bio-Rad) antibodies. Goat anti mouse or rabbit IgG Alkaline Phosphatase were purchased from Sigma-Aldrich. Images were acquired by ChemiDoc imaging systems (Bio-Rad AbD Serotech) and processed with Image Lab software (version 6.1, Bio-Rad AbD Serotec). Experiments were performed twice in duplicate.
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