Western blotting was done on six ovarian cancer tissue samples. About 20 μg of protein samples was resolved by 12% SDS-PAGE and transferred to nitrocellulose membrane (MDI) by wet transfer method using Mini Protean I (Bio-Rad Laboratories) with a constant current of 0.2 A for one hour at 4°C. Membrane was blocked with 5% bovine serum albumin in 1× PBS for one hour at room temperature. It was then washed with PBST (0.05% Tween 20 in 1× PBS) thrice for 10 minutes each. Mouse primary antibodies for alpha enolase (Santa Cruz Biotechnology) at 1:1,000 dilution was used for incubation at 4°C for 12 hours. Membrane was washed thrice with PBST and incubated with anti-mouse antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology) diluted to 1:5,000. After incubation for one hour at room temperature, it was washed with PBST and developed with diaminobenzidine (25 mg/mL) dissolved in PBS containing H2O2 (0.3% w/v). A semiquantitative analysis based on optical density was performed by ImageJ software.
Anti mouse antibody conjugated with horseradish peroxidase
Manufactured by Santa Cruz Biotechnology
Anti-mouse antibody conjugated with horseradish peroxidase is a laboratory reagent used for the detection and quantification of mouse proteins in various immunoassays. The antibody specifically binds to mouse antigens, while the horseradish peroxidase enzyme is used as a reporter molecule, allowing for colorimetric or chemiluminescent detection.
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2 protocols using anti mouse antibody conjugated with horseradish peroxidase
1
Quantitative Western Blot Analysis of Alpha Enolase in Ovarian Cancer
2
Whole Cell Lysate Preparation for p73 Analysis
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To prepare whole cell lysates, cells were collected by centrifugation and washed once with PBS and then lysed in EBC lysis buffer (Cold Spring Harb Protoc; 2006; doi:10.1101/pdb.rec8856) [50 mM Tris (pH 8.0), 120 mM NaCl, 0.5% Nonidet P-40] supplemented with aprotinin (11.5 μg/ml), leupeptin (11.5 μg/ml) phenylmethylsulfonyl fluoride (50 μg/ml), NaF (100 mM) and Na ortovanadate (0.2 mM). Protein concentration was determined by BCA following the manufacturer's instructions (G Biosciences). Proteins (25 μg) were resolved in SDS/PAGE and transferred to PVDF filters. Blots were incubated with a mouse antibody against p73 (ER-15) (Thermo-Pierce) and then incubated with an anti-mouse antibody conjugated with horseradish peroxidase (Santa Cruz). Bound antibody was detected by a chemiluminescence assay (GE Healthcare).
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