Rabbit anti ho 1 polyclonal antibody

HO-1 protein was determined by Western blot analysis. Total intracellular protein was extracted by 5X repeated freeze-thaw lysis in FT buffer (600 mM KCl, 20 mM Tris-Cl, pH 7.8, 20% glycerol, 0.4 mg/mL Pefabloc, 10 µg/mL leupeptin, 10 µg/mL pepstatin, and 5 µg/mL aprotinin). Protein concentration was determined by Bradford assay (Bio-Rad). For Western blots, 30 µg of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 4-0% gradient SDS-PAGE (Bio-Rad). Membranes were blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE, USA) for 2 h at room temperature. Primary incubation was undertaken overnight at 4℃ with a rabbit anti-HO-1 polyclonal antibody (StressGen, San Diego, Ca, USA) at 1 : 2000 and a mouse anti-β-actin antibody (Gentest) at 1 : 5000. Secondary antibody incubation was done with Alexa Fluor 680 goat anti-rabbit (Molecular Probes) and IRDye 800 goat antimouse IgG (Rockland) for 1 h at room temperature. Fluorescence was detected on an Odyssey infrared imager (LI-COR) for simultaneous detection of both species. Blot analysis was performed with the supplied Odyssey software, and HO-1 was normalized to β-actin.

HO-1 protein was determined by Western blot analysis. Total intracellular protein was extracted by 5 times repeated freeze-thaw (F-T) lysis in F-T buffer (600 mM KCl, 20 mM Tris-Cl, pH 7.8, 20% glycerol, 0.4 mg/mL Pefabloc, 10 µg/mL leupeptin, 10 µg/mL pepstatin, and 5 µg/mL aprotinin). Protein concentration was determined by Bradford assay (Bio-Rad). For western blots, 30 µg of protein was subjected to sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) on 4% SDS-polyacrylamide gels (Bio-Rad). Membranes were blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE, USA) for 2 hr at room temperature. Primary incubation was undertaken overnight at 4℃ with a rabbit anti-HO-1 polyclonal antibody (StressGen, Vancouver, CA, USA) at 1 : 2000 and a mouse anti-β-actin antibody at 1 : 5000. Secondary antibody incubation was done with Alexa Fluor 680 goat anti-rabbit and IRDye 800 goat anti mouse IgG for 1 hr at room temperature. Fluorescence was detected on an Odyssey infrared imager for simultaneous detection of both species. Blot analysis was performed with the supplied Odyssey software, and HO-1 was normalized to β-actin.