Protein samples were separated on 12% SDS-PAGE gels. After electrophoresis, the proteins were transfer to a nitrocellulose membrane (pore size 0.2 µm) with a Bio-rad Trans-Blot Cellin Towbin transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol), at 60 V, overnight. Membranes were blocked with 2% nonfat milk in PBS-T buffer (144 mg/L KH2PO4, 9 g/L NaCl, 795 mg/L Na2HPO4, pH 7.4, 0.1% Tween 20) for 1 hour. Membranes were probed with primary and secondary antibodies diluted in 2% milk in PBS-T as follows: TAg (pAb416) at 1:5,000 dilution (Harlow et al., 1986 (link)); GAPDH (Abcam ab9484) at 1:10,000; caveolin 1 (Santa Cruz SC-894) at 1:20,000; caveolin 2 (Cell signaling #8522) at 1:5,000; Clathrin heavy chain (Thermo Fisher Scientific, MA1-065) was kindly provided by Christiane Wobus (University of Michigan), used at 1:10,000, horseradish peroxidase (HRP)-conjugated ECL sheep anti-mouse (GE healthcare NA931V) at 1:5,000; and HRP-conjugated ECL donkey anti-rabbit antibody (GE healthcare NA934V) at 1:5,000. Protein bands were further visualized with HRP substrate (Millipore, WBLUF0100) and exposure to films.
Hrp conjugated ecl donkey anti rabbit antibody
Manufactured by GE Healthcare
The HRP-conjugated ECL donkey anti-rabbit antibody is a laboratory reagent used for detection and quantification of target proteins in Western blotting and other immunoassay applications. This antibody is specifically designed to bind to rabbit primary antibodies, and the horseradish peroxidase (HRP) label allows for chemiluminescent detection.
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2 protocols using hrp conjugated ecl donkey anti rabbit antibody
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Protein Samples
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Protein samples were separated by 12% SDS-PAGE. After electrophoresis, the proteins were transferred to a nitrocellulose membrane (pore size, 0.2 µm; MilliporeSigma) in Towbin transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) at 60 V overnight. Membranes were blocked with 2% nonfat milk in PBS-T buffer (144 mg/liter KH2PO4, 9 g/liter NaCl, 795 mg/liter Na2HPO4 [pH 7.4], 0.1% Tween 20) for 1 h. Membranes were probed with primary and secondary antibodies diluted in 2% milk in PBS-T as follows: anti-TAg antibody (pAb416) at a 1:5,000 dilution (93 (link)), anti-syntaxin 18 antibody (Abcam, Inc., ab156017) diluted in 5% bovine serum albumin at 1:1,000, anti-Rab18 antibody (MilliporeSigma SAB4200173) at 1:10,000, anti-VP1 antibody (pAb5G6) at 1:1000, anti-GAPDH antibody (Abcam, Inc., ab9484) at 1: 10,000, anti-ZW10 kinetochore protein antibody (Abcam, Inc., ab53676) at 1:300, anti-RAD50 interactor 1 antibody (MilliporeSigma HPA019875) at 1:200, anti-β-actin antibody (Cell Signaling 4967) at 1:10,000, horseradish peroxidase (HRP)-conjugated ECL sheep anti-mouse (GE Healthcare NA931V) at 1:5,000, and HRP-conjugated ECL donkey anti-rabbit antibody (GE Healthcare NA934V) at 1:5,000. Protein bands were visualized with HRP substrate (Millipore WBLUF0100) and exposure to X-ray film or the Syngene PXi gel doc system.
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