Each day after infection, HepG2 cells were harvested, and the total protein was extracted using NP-40 lysis buffer [50 mM Tris–HCl (pH 8.0), 0.15 M NaCl, 5 mM EDTA, 1% NP-40]. The lysates were mixed well in a rotator for 2 h at 4 °C, centrifuged at 15,000 rpm for 5 min at 4 °C, and supernatants were collected. Western blotting was performed as described previously62 (link). Membranes were incubated for 2 h at room temperature in the presence of anti-Core rabbit antibody (kindly gifted from Dr. Suzuki, Hamamatsu University School of Medicine) diluted to 1/100 or anti-Flag mouse monoclonal antibody (Merck KGaA., #4042 Darmstadt, Germany) diluted to 1/2000 with PBS-Tween. For an endogenous control, anti-GAPDH rabbit monoclonal antibody (Cell Signaling Technology, #2118, MA, USA) diluted 1/1000 were also used. After washing, membranes were incubated with peroxidase-conjugated anti-rabbit IgG (H + L) (Promega Co., #W401B, WI, USA) diluted 1/1000 or peroxidase-conjugated anti-mouse IgG (H + L) (Promega Co., #W402B) diluted 1/5000 with PBS-Tween, respectively.
Peroxidase conjugated anti rabbit igg h l
Manufactured by Promega
Sourced in United States
Peroxidase-conjugated anti-rabbit IgG (H + L) is a secondary antibody that binds to rabbit primary antibodies. It is conjugated with the enzyme peroxidase, which can be used in various immunoassay techniques to detect and quantify target proteins.
Automatically generated - may contain errors
3 protocols using peroxidase conjugated anti rabbit igg h l
1
Western Blot Analysis of HepG2 Cells
2
Detecting Bacterial T3SS Effectors
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Overnight cultures of the indicated bacterial strains were transferred into fresh LB containing 5 mM EGTA at 1% inoculum and continuously incubated at 37 °C for 3 h. The supernatants were collected and concentrated with the addition of 15% trichloroacetic acid (TCA). The samples with equivalent bacterial cells were subjected to electrophoresis on 12% SDS-PAGE. PVDF-PLUS membrane was used for the protein transfer. Rabbit antiserum specific to ExoS was used to detect T3SS effectors at 1:1000 dilution39 (link). Hybridization was performed by using 1 mg/ml peroxidase-conjugated anti-rabbit IgG (H + L) (Promega) at 1:2000 dilution, the signal was developed with ECL Plus kit (GE Health).
3
Protein Extraction and Western Blotting for HepG2 Cells
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Each day after infection, HepG2 cells were harvested, and the total protein was extracted using NP-40 lysis buffer [50 mM Tris-HCl (pH 8.0), 0.15 M NaCl, 5 mM EDTA, 1% NP-40]. The lysates were mixed well in a rotator for 2 h at 4°C, centrifuged at 15,000 rpm for 5 min at 4°C, and supernatants were collected.
Western blotting was performed as described previously 60 . Membranes were incubated for 2 h at room temperature in the presence of anti-Core rabbit antibody (kindly gifted from Dr. Suzuki, Hamamatsu University School of Medicine) diluted to 1/100 or anti-Flag mouse monoclonal antibody (Merck KGaA., #4042 Darmstadt, Germany) diluted to 1/2000 with PBS-Tween. For an endogenous control, anti-β-Actin peptide goat polyclonal antibody (Santa Cruz Biotechnology, #sc-1616, TX, USA) diluted to 1/200 or anti-GAPDH rabbit monoclonal antibody (Cell Signaling Technology, #2118, MA, USA) diluted 1/1000 were also used. After washing, membranes were incubated with peroxidase-conjugated anti-rabbit IgG (H + L) (Promega Co., #W401B, WI, USA) diluted 1/1000, peroxidase-conjugated anti-mouse IgG (H + L) (Promega Co., #W402B) diluted 1/5000, or peroxidase-conjugated anti-goat IgG (H + L) (Jackson ImmunoResearch Inc., #805-0350180, PA, USA) diluted 1/1000 with PBS-Tween, respectively.
Western blotting was performed as described previously 60 . Membranes were incubated for 2 h at room temperature in the presence of anti-Core rabbit antibody (kindly gifted from Dr. Suzuki, Hamamatsu University School of Medicine) diluted to 1/100 or anti-Flag mouse monoclonal antibody (Merck KGaA., #4042 Darmstadt, Germany) diluted to 1/2000 with PBS-Tween. For an endogenous control, anti-β-Actin peptide goat polyclonal antibody (Santa Cruz Biotechnology, #sc-1616, TX, USA) diluted to 1/200 or anti-GAPDH rabbit monoclonal antibody (Cell Signaling Technology, #2118, MA, USA) diluted 1/1000 were also used. After washing, membranes were incubated with peroxidase-conjugated anti-rabbit IgG (H + L) (Promega Co., #W401B, WI, USA) diluted 1/1000, peroxidase-conjugated anti-mouse IgG (H + L) (Promega Co., #W402B) diluted 1/5000, or peroxidase-conjugated anti-goat IgG (H + L) (Jackson ImmunoResearch Inc., #805-0350180, PA, USA) diluted 1/1000 with PBS-Tween, respectively.
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