Protein was extracted from cells in lysis buffer (50 mM Tris HCl, 1% Triton X-100, 150 mM NaCl, and 1 mM EDTA at pH 7.4) containing protease and phosphatase inhibitor mixtures (Sigma) with an ultrasonicator on ice, and cleared of cellular debris and nuclei by centrifugation at 14,000 RCF for 15 min at 4°C. 15 µg of protein per well were loaded and separated by standard 7.5% SDS-PAGE. Proteins were transferred to Immobilon-P membranes (Millipore) and then blocked with 5% dry milk for 3 h at room temperature. The blots were incubated with phospho-ERK (p-ERK) or total-ERK (t-ERK) primary antibody (Cell Signaling Technologies) overnight at 4°C and detected the following day with donkey anti-rabbit antibody conjugated to horseradish peroxidase (Jackson Immunoresearch). Signal was detected by ECL on chemiluminescent films. Phosphoprotein was normalized to the expression of the total protein on the same membrane. Densitometric analysis was performed using Image J software (NIH).
Donkey anti rabbit antibody conjugated to horseradish peroxidase
Manufactured by Jackson ImmunoResearch
Donkey anti-rabbit antibody conjugated to horseradish peroxidase is a secondary antibody that binds to rabbit primary antibodies. The horseradish peroxidase enzyme is covalently attached to the antibody, allowing for detection and visualization in various immunoassay techniques.
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Lab products found in correlation
2 protocols using donkey anti rabbit antibody conjugated to horseradish peroxidase
1
Western Blot Analysis of Phosphorylated ERK
2
Western Blot Analysis of Protein Extracts
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Protein was extracted from the cells and tissue in lysis buffer (50 mM Tris HCl, 1% Triton X-100, 150 mM NaCl, and 1 mM EDTA at pH 7.4) containing protease and phosphatase inhibitor mixtures (Sigma, St. Louis, MO) with an ultrasonicator on ice, and cleared of cellular debris and nuclei by centrifugation at 14,000 RCF for 15 min at 4°C. Fifteen micrograms of protein per well were loaded and separated by standard 7.5% or 10% SDS-PAGE. Proteins were transferred to Immobilon-P membranes (Millipore, Billerica, MA) and then blocked with 5% dry milk for 3 h at room temperature. The blots were incubated with primary antibody overnight at 4°C and detected the following day with donkey anti-rabbit antibody conjugated to horseradish peroxidase (Jackson Immunoresearch, West Grove, PA). Signal was detected by ECL on chemiluminescent films. Densitometric analyses were performed with Image J software (NIH, Bethesda, MD).
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