Western blot analyses were performed as previously described (25 (link)). We used the following primary antibodies (See Supplementary Table 1 ). The nitrocellulose-bound primary antibodies, were detected with anti–rabbit IgG horseradish peroxidase–linked antibody or anti–mouse IgG horseradish peroxidase–linked antibody (EMD Millipore; Temecula, CA USA), and were detected by the enhanced chemoluminescence staining ECL (GE Healthcare–Amersham Pharmacia, Buckinghamshire, U.K.).
Anti rabbit igg horseradish peroxidase linked antibody
Manufactured by Merck Group
Sourced in United States, United Kingdom
Anti–rabbit IgG horseradish peroxidase–linked antibody is a lab equipment product that functions as a detection reagent. It is used to identify and quantify the presence of rabbit immunoglobulin G (IgG) in biological samples through a color-based enzymatic reaction.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse igg horseradish peroxidase linked antibody, by Merck Group (2 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Amersham ecl detection reagent, by GE Healthcare (1 mentions)
Amersham ecl, by Cytiva (1 mentions)
Mse soniprep 150, by Sanyo (1 mentions)
Nitrocellulose transfer membrane, by Cytiva (1 mentions)
Nupage mes sds running buffer, by Thermo Fisher Scientific (1 mentions)
Nupage bis tris mini gel, by Thermo Fisher Scientific (1 mentions)
Ecl detection system, by GE Healthcare (1 mentions)
Whatman, by Cytiva (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
A2066, by Merck Group (1 mentions)
A0545, by Merck Group (1 mentions)
L2170, by Merck Group (1 mentions)
4 protocols using anti rabbit igg horseradish peroxidase linked antibody
1
Western Blot Protein Detection
2
Immunoblot Analysis Protocol
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Immunoblot analyses were performed as previously described61 (link). Primary antibodies are listed in Supplementary Table 3 with their respective concentrations. Nitrocellulose-bound primary antibodies were detected with anti-rabbit IgG horseradish peroxidase-linked antibody or anti-mouse IgG-horseradish peroxidase-linked antibody (EMD-Millipore) and detected by Amersham ECL detection reagent (GE Healthcare).
3
Western Blotting of Bacterial Proteins
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Bacterial samples required for Western blotting were grown aerobically overnight in M9 minimal medium at 37°C. The following day cultures were subcultured and grown in M9 minimal medium at 37°C to approximately mid‐logarithmic growth phase (OD600nm ~0.6) then harvested by centrifugation, and cell pellets were re‐suspended in 50 mM Tris–HCl (pH 8.0). Protein extracts were prepared by sonication on ice with an MSE Soniprep 150 (Sanyo, UK) for four pulses of 30 s with a 30‐s pause between each pulse. A Bradford assay was carried out to quantify the protein concentration, and 10 μg of protein was run on 4–12 % NuPAGE® Bis‐Tris mini gels with NuPAGE® MES SDS running buffer (Life Technologies, UK). Protein was transferred to nitrocellulose transfer membranes (Whatman, UK), and analyzed by Western blotting using AcrB antibody at a 1 : 1,000 dilution. Blots were developed using anti‐rabbit IgG horseradish peroxidase‐linked antibody (Sigma, UK) at a 1 : 25,000 dilution and analyzed using the ECL detection system (GE Healthcare UK).
4
Western Blot Analysis of Lung Membranes and HEK293 Cells
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Isolated lung membranes or HEK293 cell lysate were heated to 95 °C in Laemmli sample buffer (Bio-Rad, Jerusalem, Israel) for 5 minutes. Lysates were resolved by 4–12% Bis-Tris (for lung membranes) or 3–8% Tris-Acetate sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, for HEK293 cell lysate) (Invitrogen, Carlsbad, CA, USA) and transferred onto nitrocellulose membranes (Invitrogen). Membranes were blocked with 5% nonfat blotting grade blocker (170–6404; BioRad, Hercules, CA, USA) in tris-buffered saline/Tween-20 for 1 hour at room temperature. For immunoblotting of lung cells-membranes, the membranes were incubated with purified ricin or RCA (20 µg/ml) for 2 hours, washed and incubated with rabbit-anti-ricin polyclonal antibody for 2 hours. For immunoblotting of HEK293 cell lysate, the membranes were incubated with rabbit anti-LRP1 C terminal antibody (L2170, Sigma–Aldrich) and rabbit anti-actin (A2066, Sigma-Aldrich) over night at 4 °C. All membranes were incubated with anti-rabbit IgG horseradish peroxidase-linked antibody (A0545, Sigma-Aldrich) for 1 hour, developed and band intensities were quantified as previously described74 .
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