All western blots were performed by transferring samples from SDS-PAGE gels to nitrocellulose membranes at 1.00A for 36 min. Membranes were blocked in 5% milk in TBS/T plus 0.02% sodium azide. The following primary antibodies were used – mouse monoclonal anti-MBP-HRP 1:1000 (NEB), mouse monoclonal anti-FLAG-HRP 1:4000 (Sigma), rabbit polyclonal anti-AKAP79 C-terminus (Millipore), rabbit polyclonal anti-PP2B A subunit (Millipore), mouse monoclonal anti-PP2B B subunit (Abcam), and rabbit polyclonal anti-CaM (Santa Cruz). Blots were incubated in primary antibody dilutions overnight at 4˚C, and then washed 3 times for 5 min in TBS/T. Blots were then incubated with the appropriate secondary antibody conjugated to HRP at 1:10,000 dilution for 1 hr at room temperature. After three more TBS/T washes, blots were developed and imaged.
Mouse monoclonal anti flag hrp
Manufactured by Merck Group
The Mouse monoclonal anti-FLAG-HRP is a laboratory reagent used for the detection of proteins tagged with the FLAG peptide epitope. It is an antibody that is conjugated with horseradish peroxidase (HRP), which allows for colorimetric or chemiluminescent detection of the tagged proteins.
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2 protocols using mouse monoclonal anti flag hrp
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Antibody-based protein detection protocol
2
Coimmunoprecipitation of KLF4 and Runx2 in 293T Cells
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After 293T cells were transfected with Flag-KLF4 and HA-Runx2 for 48 h, the cells were washed with chilled PBS and lysed in extraction buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, PMSF, and protease inhibitors). Cell lysates were immunoprecipitated with mouse monoclonal anti-HA antibody (Roche). Cell lysates or immunoprecipitated samples were subsequently separated by SDS-PAGE and transferred to PVDF membranes (EMD Millipore). The membranes were blocked with TBS-T (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) containing 5% skim milk and probed with HRP-conjugated antibodies including mouse monoclonal anti–Flag-HRP (Sigma-Aldrich) and mouse monoclonal anti–HA-HRP (Sigma-Aldrich). Signals were detected with enhanced chemiluminescence and analyzed by use of a luminescent image analyzer (LAS3000; Fujifilm).
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