Secondary antibodies conjugated to horseradish peroxidase

Manufactured by Cytiva

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Secondary antibodies conjugated to horseradish peroxidase are detection reagents used in immunoassays and immunohistochemistry. They bind to the primary antibody and catalyze a colorimetric or chemiluminescent reaction, enabling the visualization and quantification of the target analyte.

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Lab products found in correlation

Polyvinylidene difluoride membrane, by Merck Group (1 mentions) Biomax xar film, by Kodak (1 mentions) Semi dry transfer cell, by Bio-Rad (1 mentions) Human bcl 2, by Santa Cruz Biotechnology (1 mentions) Enhanced chemiluminescence method, by Cytiva (1 mentions) Hybond p pvdf membrane, by Cytiva (1 mentions) β actin ac 15, by Merck Group (1 mentions) Hybond, by Cytiva (1 mentions)

Spelling variants of this product

Horseradish peroxidase (66 citations) Secondary horseradish peroxidase-conjugated antibodies (6 citations)

3 protocols using secondary antibodies conjugated to horseradish peroxidase

Western Blot Analysis of Apoptosis-Related Proteins

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Protein samples obtained as described were subjected to 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) using a semidry transfer cell (Bio-Rad). Membranes were then blocked for 1 h at room temperature with Tris-buffered saline-Tween 20 (TBS-T; 20 mM Tris-HCl (pH 7.4), 137.5 mM NaCl, and 0.1% Tween 20) containing 5% skim milk, washed with TBS-T three times, and probed at 4°C for 12 h with rabbit antibodies to sheep cytochrome c (Sigma-Aldrich), human Bcl-2 (1 : 500 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), and human Bcl-2-associated X protein (Bax; 1 : 500 dilution; Santa Cruz Biotechnology) or with goat antibodies to human β-actin (1 : 500 dilution; Santa Cruz Biotechnology). Subsequently, the membranes were washed three times with TBS-T, labeled for 1 h at room temperature with appropriate secondary antibodies conjugated to horseradish peroxidase (1 : 2000 dilution; Amersham Biosciences, Piscataway, NJ, USA), washed, stained with West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA), and visualized on X-ray film (KODAK BioMax XAR Film; KODAK, Rochester, NY, USA).

Iguchi M., Hiroi M., Kanegae H, & Ohmori Y. (2018). Costimulation of Murine Osteoblasts with Interferon-γ and Tumor Necrosis Factor-α Induces Apoptosis through Downregulation of Bcl-2 and Release of Cytochrome c from Mitochondria. Mediators of Inflammation, 2018, 3979606.

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Quantitative Immunoblotting Analysis

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The procedure for immunoblotting has been previously described (Ke and Gibson, 2004 (link); Wang et al., 2007a (link)). Briefly, aliquots of proteins (30 μg) were loaded into the lanes of a sodium dodecyl sulfate-polyacrylamide gel. The proteins were separated by electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 5% BSA in 0.01 M Tris-buffered saline (TBS) (pH 7.4) and 0.05% Tween-20 (TBST) at room temperature for 1 h. Then, the membranes were incubated with primary antibodies directed against target proteins overnight at 4 °C. The final dilutions for primary antibodies were: GRP78, 1:1,000; XBP-1, 1:1,000; p-eIF2α, 1:1,000; Chop, 1:500; caspase-12, 1:1,000; HNE, 1:1,000; DNP, 1:1,000; and actin, 1:5,000. After two quick washes in TBST, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (Amersham, Arlington, Heights, IL) diluted at 1:5000 in TBST containing 5% BSA for 1 h. The immunocomplexes were detected by the enhanced chemiluminescence method (Amersham). The density of immunoblotting was quantified with the software of ImageJ (version 1.48; National Institutes of Health, Bethesda, MD).

Wang X., Xu M., Frank J.A., Ke Z.J, & Luo J. (2017). Thiamine deficiency induces endoplasmic reticulum stress and oxidative stress in human neurons derived from induced pluripotent stem cells. Toxicology and applied pharmacology, 320, 26-31.

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Western Blot Analysis of HSP72 and β-Actin

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The cells were harvested from flasks, and lysed in a lysis buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 1 mM MgCl₂, 2.5 mM VO₄, 1 mM PMSF, 2.5 mM EDTA, 0.5% Triton X-100, 0.5% NP-40, 5 μg/mL of aprotonin, peptatin A, and leupeptin) for 60 min. Protein samples (500 μg) were separated on 10% or 12% SDS–PAGE gels, and transferred to Hybond-P PVDF membranes (Amersham Biosciences, Stockholm, Sweden). After blocking with 5% non-fat dry mild in TBS-T buffer (20 mM Tris, pH 7.6, 100 mM NaCl, 0.1% Twn-20) for 2 h at room temperature, the membranes were probed with a 1:1000 dilution of anti-HSP72 (DO-1, Santa Cruz Biotechnology, CA, USA) overnight at 4 °C, and a 1:5000 dilution of β-actin (AC-15, Sigma–Aldrich) to correct for differences in protein loading, followed by incubation in a 1:2000 dilution of secondary antibodies conjugated to horseradish peroxidase (Amersham Biosciences) for 1 h at room temperature. Protein bands were detected using ECL detection (Amersham Biosciences). All of the Western immunoblots were performed at least three times.

Lin K.C., Lin H.J., Chang C.P, & Lin M.T. (2015). Decreasing or increasing heat shock protein 72 exacerbates or attenuates heat-induced cell death, respectively, in rat hypothalamic cells. FEBS Open Bio, 5, 724-730.

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