Cells were homogenized in the presence of RIPA buffer (Cell Signaling) with protease inhibitors (Thermo Scientific, Rockford, IL). Thereafter, cell lysates were centrifuged to remove any residual debris. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Heat-denatured and β-mercaptoethanol-reduced samples (20μg protein/lane) were electrophoresed on 4–20% BisTris–SDS gradient gels (Invitrogen). After electrophoresis, proteins were transferred into a polyvinylidene fluoride membrane, and blocked with casein-blocking buffer (Sigma). The blocked membrane was incubated in rabbit anti-TBM antibody (Abcam, Cambridge, MA), followed by goat anti-rabbit IgG-horseradish peroxidase antibody (Thermo Scientific). As a control, mouse anti-β-actin was used at a dilution of 1:500 for the primary antibody, followed by anti-mouse IgG horseradish peroxidase antibody (Santa Cruz, Dallas, Tx). Positive signals were detected with Pierce ECL Western Blotting Substrate (Thermo Scientific) and photographic imaging.
Goat anti rabbit igg horseradish peroxidase antibody
Manufactured by Thermo Fisher Scientific
The Goat anti-rabbit IgG-horseradish peroxidase antibody is a secondary antibody that binds to rabbit immunoglobulin G (IgG) and is conjugated to the enzyme horseradish peroxidase. This antibody can be used in various immunoassay and detection techniques to identify the presence of rabbit IgG in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Casein blocking buffer, by Merck Group (1 mentions)
Las 4000 mini luminescent image analyzer, by Fujifilm (1 mentions)
Supersignal west femto system, by Thermo Fisher Scientific (1 mentions)
2 protocols using goat anti rabbit igg horseradish peroxidase antibody
1
Western Blot Analysis of TBM Protein
2
Immunoblotting of CAP1 in SFVmfu-Infected Cells
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Whole cell lysates of TE671 cells were obtained with RIPA buffer (50 mM Tris-Cl [pH 7.4], 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% aprotinin, 50 mM NaF, and 0.1 mM Na3VO4) 48 or 96 h after the mock or SFVmfu infection (at an MOI of 0.34). Similarly, whole cell lysates of uninfected TE671 and TE671/SFVmfu(PI) cells were obtained with RIPA buffer. Lysates were mixed with Laemmli sample buffer supplemented with 2-mercaptoethanol and boiled at 95°C for 5 min. Each boiled sample was electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel and electrically blotted onto a polyvinylidene difluoride membrane. Each blotted membrane was blocked in 5% milk/Tris-buffered saline with Tween 20 and incubated with an anti-CAP1 antibody (Abcam [catalog number: EPR8339{B}]) as the primary antibody or a goat anti-rabbit IgG-horseradish peroxidase antibody (Thermo Fisher Scientific [catalog number: 31466]) as the secondary antibody. Antibody reactions were detected and processed with the SuperSignal West Femto system (Thermo Fisher Scientific) using a Luminescent Image Analyzer LAS4000 mini (Fujifilm).
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