Cultured cells were harvested and resuspended in lysis buffer (20 mM Tris/Cl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40, and 2% SDS). Protein concentration of cleared lysates was determined using the Bio-Rad protein assay reagent. 25 μg of total protein were separated by denaturing PAGE and subjected to Western blotting using mouse monoclonal anti-FLAG antibody (M2; Sigma-Aldrich, 1:1,000) followed by probing with an HRP-coupled anti-mouse light chain–specific secondary antibody (1:10,000; Jackson Immuno Research). Detection occurred by using Clarity Western ECL substrate and a ChemiDoc Touch Imaging System (Bio-Rad). After stripping, the membrane was re-probed using mouse anti–α-tubulin antibody (DM1A; Sigma-Aldrich, 1:1,000).
Hrp coupled anti mouse light chain specific secondary antibody
Manufactured by Jackson ImmunoResearch
HRP-coupled anti-mouse light chain–specific secondary antibody is a laboratory reagent used to detect and quantify mouse immunoglobulins in various immunoassays. It is conjugated with horseradish peroxidase (HRP), an enzyme that can be used for colorimetric or chemiluminescent detection.
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2 protocols using hrp coupled anti mouse light chain specific secondary antibody
1
Western Blot Analysis of FLAG-Tagged Proteins
2
Western Blot Analysis of FLAG-Tagged Proteins
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Cultured cells were harvested and resuspended in lysis buffer (20 mM Tris/Cl pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 2% SDS). Protein concentration of cleared lysates was determined using the BioRad protein assay reagent. 25 µg of total protein were separated by denaturing PAGE and subjected to Western blotting using mouse monoclonal anti-FLAG antibody (M2, Sigma Aldrich, 1:1000) followed by probing with an HRP-coupled anti-mouse light chain-specific secondary antibody (1:10000, Jackson Immuno Research). Detection occurred by using Clarity Western ECL substrate and a ChemiDoc Touch Imaging System (BioRad). After stripping, the membrane was re-probed using mouse anti-alpha-tubulin antibody (DM1A, Sigma Aldrich, 1:1000).
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