Dimethyl fumarate (DMF), lypopolysaccharide (from E. Coli serotype 0127:B8) and all other reagents were purchased from Sigma Aldrich (France) unless otherwise specified. CORM-401 and ethyl prop-2-yn-1-yl fumarate (EPF) were synthesized in our laboratories as previously described [26] (link), [27] (link), [28] (link), [30] (link). RPMI-1640 medium, fetal bovine serum (FBS) and L -glutamine were from Lonza, while penicillin, streptomycin and Dulbecco Phosphate Buffer Solution (DPBS) were purchased from Life Technologies (Aubin, France). Antibodies were purchased from Enzo Life Sciences (HO-1 rabbit polyclonal), Cell Signaling Technology (β-actin mouse monoclonal) and Santa Cruz Biotechnology (Nrf2 clone C-20 rabbit polyclonal and Lamin A/C clone N-18 goat polyclonal). Nuclear Extract Kit for the isolation and preparation of nuclear extracts was from Active Motif (Paris, France). The CO sensitive probe COP-1 was kindly provided by Prof. Christopher Chang from the University of California, Berkeley [31] (link).
Ho 1 rabbit polyclonal
Manufactured by Cell Signaling Technology
HO-1; rabbit polyclonal is a lab equipment product from Cell Signaling Technology. It is a rabbit-derived polyclonal antibody that targets the Heme Oxygenase-1 (HO-1) protein.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Sod2 rabbit monoclonal, by Cell Signaling Technology (2 mentions)
Ponceau s staining, by Merck Group (2 mentions)
Mouse monoclonal β actin, by Santa Cruz Biotechnology (1 mentions)
Rpmi 1640 medium, by Lonza (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Dimethyl fumarate, by Merck Group (1 mentions)
Dulbecco phosphate buffer solution, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Lonza (1 mentions)
L glutamine, by Lonza (1 mentions)
3 protocols using ho 1 rabbit polyclonal
1
Dimethyl Fumarate and Oxidative Stress
2
Western Blot Analysis of Antioxidant Proteins
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Samples boiled in 6x Laemmli buffer were subjected to standard SDS-PAGE (10%) and electrophoretically blotted onto Immobilon-P polyvinylidene difluoride membranes (Merck Millipore, Ltd., UK). Total protein was quantified using Ponceau S staining (Merck Millipore, Ltd., UK) and membranes were blotted using antibodies raised against murine haem oxygenase-1 (HO-1; rabbit polyclonal, 1 : 1000, Cell Signaling Technology, Leiden, The Netherlands) or superoxide dismutase 2 (SOD2; rabbit monoclonal, 1 : 1000, Cell Signaling Technology, Leiden, The Netherlands) in Tris-buffer saline solution containing 0.1% Tween-20 and 5% (w/v) nonfat dry milk overnight at 4°C. Membranes were washed with Tris-buffer saline solution containing 0.1% Tween-20 and incubated with secondary antibody (horseradish peroxidase–conjugated goat anti-rabbit 1 : 5000; Thermo Fisher Scientific, UK) for 90 min at room temperature. Proteins were then detected using enhanced chemiluminescence detection (2.5 mM luminol, 0.4 mM p-coumaric acid, 7.56 mM H2O2 in 1 M Tris, pH 8.5) and visualised on X-ray film (Scientific Laboratory Supplies Limited, Nottingham, UK). Films were digitized and analysed using ImageJ 1.51w software (National Institutes of Health).
3
Protein Expression Quantification via Western Blot
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Samples boiled in 6× Laemmli buffer were subjected to standard SDS-PAGE (10%) and electrophoretically blotted onto Immobilon-P polyvinylidene difluoride membranes (Merck, UK). Total protein was quantified using Ponceau S staining (Merck, UK) and membranes were blotted using antibodies raised against murine haem oxygenase-1 (HO-1; rabbit polyclonal, 1:1000, Cell Signaling Technology, Leiden, The Netherlands) or superoxide dismutase 2 (SOD2; rabbit monoclonal, 1:1000, Cell Signaling Technology, Leiden, The Netherlands) in Tris-buffer saline solution containing 0.1% Tween-20 and 5% (w/v) non-fat dry milk overnight at 4 o C. Membranes were washed with Tris-buffer saline solution containing 0.1% Tween-20, and incubated with secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit 1:5,000; ThermoFisher Scientific, UK), for 90 min at room temperature. Proteins were then detected using enhanced chemiluminescence detection (2.5 mM luminol, 0.4 mM p-coumaric acid, 7.56 mM H 2 O 2 in 1 M Tris, pH 8.5) and visualised on X-ray film (Scientific Laboratory Supplies Limited, Nottingham, UK). Films were digitized and analysed using ImageJ 1.51w software (National Institutes of Health).
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