To analyze the expression of HO-1, BMMNCs from WT and PLC-β2-deficient mice were harvested, centrifuged, and washed twice with PBS. Protein extracts were then obtained after cell lysis using RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Santa Cruz Biotechnology) and centrifugation at 15,000 rpm at −4 °C for 15 min. The protein concentrations were measured with the Pierce BCA Protein Assay Kit (Pierce, Rockford, IL) and Multimode Analysis Software (Beckman Coulter). Next, adjusted protein lysates (80 μg per sample) were separated on a 4–12 % SDS-PAGE gel, and fractionated proteins were transferred to a PVDF membrane (Bio-Rad). After blocking with 2.5 % non-fat dry milk in Tris-buffered saline containing 0.1 % Tween (TBST) for 1 h at room temperature, then washing with TBST, the membranes were incubated with a rabbit anti-HO-1 polyclonal antibody (Enzo Life Sciences, NY, USA, diluted 1:1000) overnight at 4 °C. Equal loading of proteins in all lanes was assured by reprobing with rabbit anti-β-actin monoclonal antibody (Novus Biologicals, USA, diluted 1:1000). Enhanced chemiluminescence (ECL) reagent (Amersham Life Sciences) and film (Hyperfilm, Amersham Life Sciences) were used for band visualization [19 (link), 21 (link)].
Rabbit anti β actin monoclonal antibody
Manufactured by Novus Biologicals
Sourced in United States
Rabbit anti-β-actin monoclonal antibody is a laboratory reagent used for the detection and quantification of the β-actin protein, which is a ubiquitous cytoskeletal protein found in all eukaryotic cells. This antibody can be used in various immunoassay techniques, such as Western blotting, immunohistochemistry, and flow cytometry, to analyze the expression levels of β-actin in biological samples.
Automatically generated - may contain errors
3 protocols using rabbit anti β actin monoclonal antibody
1
Western Blot Analysis of HO-1 Expression
2
Lipid Signaling Regulates HO-1 Expression
3
Heme Oxygenase-1 Expression in Cell Lines
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Cells (CRL5853, CRL2062) were cultured with FSH (1 mU/ml), LH (1 mU/ml), or PRL (0.5 µg/ml) in serum-free RPMI-1640 medium for 6 h at 37°C. The harvested cells were centrifuged and washed with ice-cold PBS. The total protein extracts were collected, and their concentrations were then measured. The concentration-adjusted extracted proteins (70 µg/each sample) were then separated on a 4–12% SDS-PAGE gel and then transferred to a PVDF membrane. Next, the membranes were blocked with 2.5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween (TBST) for 1 h at room temperature. After washing with TBST, the membranes were incubated with rabbit anti-HO-1 polyclonal antibody (diluted at 1:1,000; Enzo Life Sciences, Inc., Farmingdale, NY, USA) overnight at 4°C. To assure equal protein loading in all the lanes, the blots were then reprobed with rabbit anti-β-actin monoclonal antibody (diluted at 1:1,000; Novus Biologicals, Littleton, CO, USA). All membranes were treated with ECL reagent, and subsequently exposed to film for band visualization.
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