All yeast strains were grown in iron limited medium for 24 hours at 30 1C. Cell pellets were collected by centrifugation at 2000 Â g at 4 1C for 10 min, washed twice with ice-cold water and lysed with glass beads (lysis buffer: 50 mM HEPES pH 7.5, 150 mM NaCl, 2.5 mM EDTA, 1% v/v Triton X100 and freshly added protease inhibitor). After lysis, membranes were collected by centrifugation at 18000 Â g (4 1C, 30 min). Samples were re-suspended in SDS loading buffer (0.5 M Tris-HCl, pH 6.8, 10% SDS, 0.5% (w/v) bromophenol blue, 87% glycerol, 100 mM DTT) and 50 mg of membranes were loaded on a 4-12% Bis-Tris gel (Invitrogen) and blotted onto PVDF membranes. ATP7B and Atox1 were detected with monoclonal rabbit ATP7B and Atox1 antibodies, respectively (Abcam, 1 : 1000 dilution), upon incubation overnight, 4 1C. Next, blots were incubated with horseradish peroxidase conjugated anti-rabbit IgG reagent (Thermo Scientific Pierce) for 15 min, 4 1C. Bands were detected by Piercet Fast Western Blot Kits, SuperSignalt West Femto, Rabbit (Thermo Scientific Pierce) and visualized with a BioRad ChemiDoc XRS image analyzer.
Horseradish peroxidase conjugated anti rabbit igg reagent
Manufactured by Thermo Fisher Scientific
The Horseradish peroxidase conjugated anti-rabbit IgG reagent is a laboratory product designed for use in various immunoassays. It consists of anti-rabbit IgG antibodies that are conjugated to the enzyme horseradish peroxidase. This reagent can be used to detect and quantify the presence of rabbit immunoglobulin G (IgG) in a sample.
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Lab products found in correlation
3 protocols using horseradish peroxidase conjugated anti rabbit igg reagent
1
Membrane Protein Analysis in Iron-Starved Yeast
2
Membrane Protein Isolation and Detection
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All yeast strains were grown in iron limited medium for 30 h at 30 °C. Cells were spun down by centrifugation at 20009g at 4 °C for 10 min. Cells pellets were washed twice with ice-cold water and resuspended in lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 2.5 mM EDTA, 1% v/v Triton X100 and freshly added protease inhibitor). After disruption with glass beads, membranes were collected by centrifugation at 18,0009g (4 °C, 30 min). Samples were re-suspended in SDS loading buffer (0.5 M Tris-HCl, pH 6.8, 10% SDS, 0.5% (w/v) bromophenol blue, 87% glycerol, 100 mM DTT) and 50 mg of membranes were loaded on a 4-12% Bis-Tris gel (Invitrogen) and blotted onto PVDF membranes. ATP7B and Atox1 were detected with monoclonal rabbit ATP7B and Atox1 antibodies, respectively (Abcam, 1:1000 dilution), upon incubation overnight at 4 °C. Next, blots were incubated with horse radish peroxidase conjugated anti-rabbit IgG reagent (Thermo Scientific Pierce) for 15 min, 4 °C. Bands were detected by Pierce TM Fast Western Blot Kits, SuperSignal TM West Femto, Rabbit (ThermoScientific Pierce) and visualized with a BioRad ChemiDoc XRSimage analyzer.
3
Western Blot Analysis of Yeast ATP7B Variants
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Yeast was grown in iron-limited medium for 30 h at 30°C. Cells were spun down by centrifugation at 2000 × g at 4°C for 10 min. Cells’ pellets were washed twice with ice-cold water and resuspended in lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 2.5 mM EDTA, 1% v/v Triton X-100, and freshly added protease inhibitor). After disruption with glass beads, membranes were collected by centrifugation at 18,000 × g (4°C, 30 min). Samples were resuspended in SDS loading buffer (0.5 M Tris-HCl (pH 6.8), 10% SDS, 0.5% (w/v) bromophenol blue, 87% glycerol, and 100 mM DTT), and 50 mg of membranes were loaded on a 4–12% Bis-Tris gel (Invitrogen, Carlsbad, CA) and blotted onto PVDF membranes. ATP7B variants were detected with monoclonal rabbit ATP7B antibodies (1:1000 dilution; Abcam, Cambridge, UK) upon incubation overnight, followed by incubation with horseradish-peroxidase-conjugated anti-rabbit IgG reagent (Thermo Scientific Pierce, Waltham, MA) for 15 min at 4°C. Bands were visualized by Pierce Fast Western Blot Kits, SuperSignal West Femto, Rabbit (Thermo Scientific Pierce) in a Bio-Rad ChemiDoc analyzer (Bio-Rad, Hercules, CA).
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