Peroxidase conjugated secondary antibody

Manufactured by Jackson ImmunoResearch

Sourced in United States, Cameroon, United Kingdom, Germany

Peroxidase-conjugated secondary antibodies are a type of laboratory reagent used in various immunoassays. They consist of a secondary antibody that is chemically linked to the enzyme horseradish peroxidase. This conjugate is used to detect and amplify the signal from a primary antibody that has bound to its target antigen.

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Lab products found in correlation

Clarity western ecl substrate, by Bio-Rad (8 mentions) Chemidoc touch imaging system, by Bio-Rad (7 mentions) Ripa buffer, by Thermo Fisher Scientific (6 mentions) Bca protein assay kit, by Thermo Fisher Scientific (6 mentions) Anti β actin, by Merck Group (6 mentions) Ecl detection system, by Cytiva (6 mentions) Nitrocellulose membrane, by Cytiva (6 mentions) Pvdf membrane, by Merck Group (5 mentions) Anti flag m2, by Merck Group (5 mentions) Ripa buffer, by Merck Group (5 mentions) Polyvinylidene fluoride membrane, by Merck Group (5 mentions) Multi gauge software, by Fujifilm (4 mentions) Anti ha, by Merck Group (4 mentions) β actin, by Santa Cruz Biotechnology (4 mentions) Anti myc, by Merck Group (4 mentions) Nupage bis tris polyacrylamide gel, by Thermo Fisher Scientific (3 mentions) Supersignal west substrate, by Thermo Fisher Scientific (3 mentions) Runx2, by Cell Signaling Technology (3 mentions) Nitrocellulose membrane, by Bio-Rad (3 mentions) Anti tubulin, by Merck Group (3 mentions)

Close spelling variants of this product

Horseradish peroxidase (HRP)-conjugated secondary antibodies (193 citations) Peroxidase-conjugated bovine anti-mouse IgG secondary antibody (23 citations) Secondary antibody conjugated to horseradish peroxidase (17 citations) Peroxidase-conjugated goat anti-rabbit secondary antibody (16 citations) Anti-rabbit peroxidase-conjugated secondary antibody (10 citations) Horseradish peroxidase-conjugated goat anti-mouse secondary antibody (8 citations) Anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (7 citations) Peroxidase-conjugated goat anti-mouse secondary antibody (7 citations) Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies (5 citations) Horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (5 citations)

152 protocols using peroxidase conjugated secondary antibody

Protein Detection and Luminescence Assays

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Western blotting was performed following standard procedure. The primary antibody for V5-tag (Abcam, Shanghai, China) and peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, USA) were used for targets detection. The immunoreactive proteins were detected by SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).
Luciferase assay was performed in the white, opaque 96-well plates using 20 μL of culture supernatant for each well, and 50 μL/well Gluc buffer (100 mM Tris-HCl at pH 8.0, 10 μM coelenterazine, 0.3% Triton X-100, and 70 mM NaI) or Cypridina Glow Assay Buffer containing 1 × Vargulin (Thermo Fisher Scientific) was added for Gluc or hCluc assay. Total chemiluminescence was acquired using the SpectraMax i3x multi-mode microplate reader (Molecular Devices). Fluorescence intensities in cells were measured using the same microplate reader.

Bei Z.C., Yu H., Wang H., Li Q., Wang B., Zhang D., Xu L., Zhao L., Dong S, & Song Y. (2023). Orthogonal dual reporter-based gain-of-signal assay for probing SARS-CoV-2 3CL protease activity in living cells: inhibitor identification and mutation investigation. Emerging Microbes & Infections, 12(1), 2211688.

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Western Blot Protein Detection with GSI Treatment

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Cells treated with or without GSI at various doses were lysed in Lysis buffer (20 mM Tris pH 7.5/150 mM NaCl/12 mM EDTA/10% glycerol/1% Triton X-100) containing a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN) and PhosphoStop (Roche Applied Science, Indianapolis, IN). The protein concentration was measured using a Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Fifty μg of protein was mixed with an equal volume of 2x SDS loading buffer (Invitrogen, Carlsbad, CA) and boiled for 5 min before applying the sample to SDS-PAGE. After SDS-PAGE, the protein gel was blotted on a nitrocellulose membrane Hybond C extra (Amersham Biosciences, Pittsburgh, PA) using a Bio-Rad blotting apparatus (BioRad, Hercules, CA) for one hour. The blot was blocked with 5% dry milk in TBST for one hour, washed twice in TBST, and then incubated with antibodies (1∶1000) in 5% BSA at 4°C overnight. The blot was washed three times in TBST and then incubated with peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) in 5% dry milk at room temperature for 1 hour. After washing three times in TBST, the blot was incubated for 5 minutes with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and X-ray film (Denville Scientific, Metuchen, NJ) was then developed.

Abel E.V., Kim E.J., Wu J., Hynes M., Bednar F., Proctor E., Wang L., Dziubinski M.L, & Simeone D.M. (2014). The Notch Pathway Is Important in Maintaining the Cancer Stem Cell Population in Pancreatic Cancer. PLoS ONE, 9(3), e91983.

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Immunoblotting Analysis of Caveolin-1 in Hyperosmotic Stress

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As described previously (Reppetti et al., 2019 (link)), control cells and hyperosmolarity-treated cells were collected in a lysis buffer. Then, protein concentration was determined using the commercial Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, United States).
For immunoblotting studies, 50 μg proteins from each sample of the lysate of Swan 71 cells were loaded and resolved on a 12.5% polyacrylamide gel and onto nitrocellulose membranes (Hybond ECL, Amersham Pharmacia Biotech Ltd., Pittsburgh, PA, United States). After blocking, membranes were rinsed and incubated overnight with an anti-Cav-1 (1:1,000; Santa Cruz Biotechnology Incorporation, CA, United States) followed by incubation with a peroxidase-conjugated secondary antibody (1:10,000; Jackson Immuno Research Laboratories, Incorporation, West Grove, PA, United States). Immunoreactivity was detected using the enhanced chemiluminescence (ECL) detection reagent (Amersham ECL plus, GE Healthcare Life Sciences, Pittsburgh, PA, United States). Bands were quantified by densitometric analysis using the ImageJ 1.51r^® software package (Bethesda, MD, United States). Results were expressed as the relative abundance of each protein and we used Ponceau staining as a loading control (Gilda and Gomes, 2013 (link)).

Reppetti J., Medina Y., Farina M., Damiano A.E, & Martínez N.A. (2021). Hyperosmolarity Impairs Human Extravillous Trophoblast Differentiation by Caveolae Internalization. Frontiers in Physiology, 12, 760163.

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Evaluating Protein Signaling in Cancer

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Western blotting (WB) was conducted to evaluate the protein levels in cancer tissues and lung cancer cells. Protein concentrations were determined using the BCA Protein Assay Kit (Thermo, MA, USA). Proteins were detected using primary antibodies against (Cell Signaling Technology) p-JNK, JNK, pERK1/2, ERK1/2, p-p38, p38, TLR4, MyD88, IκBα, p65, β-actin, and (Abcam, Cambridge, MA) IKKα and IKKβ. Peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, USA) was used and immunoreactive bands were visualized based on the Enhanced Chemiluminescence (ECL) detection system (Thermo, MA, USA). The phosphorylation level of the selected protein was expressed as the ratio of the phosphorylated protein to total protein.

Zhao B., Hui X., Jiao L., Bi L., Wang L., Huang P., Yang W., Yin Y., Jin S., Wang C., Zhang X, & Xu L. (2020). A TCM Formula YYWY Inhibits Tumor Growth in Non-Small Cell Lung Cancer and Enhances Immune-Response Through Facilitating the Maturation of Dendritic Cells. Frontiers in Pharmacology, 11, 798.

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Protein Expression Analysis of DHA-treated Cells

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Cells with the treatment of DHA were harvested and rinsed with PBS, then extracted in 300 μl radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China). Proteins were determined by the bicinchoninic acid assay (BCA) kits (Beyotime, Shanghai, China). The proteins were loaded and size-fractionated by SDS-PAGE gels, then transferred onto PVDF membranes (EMD Millipore, Billerica, MA, U.S.A.). After blocking, the membranes were incubated with primary antibodies at 4°C overnight, including anti-GPX4 (1:1000), ACSL4 (1:1000), xCT (1:1000), HO-1 (1:1000), TfR (1:500) and β-actin (1:5000) or GAPDH (1:5000). All these antibodies were purchased from Abcam (Cambridge, MA, U.S.A.). After TBST washes, the membranes were incubated with the appropriate peroxidase conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, U.S.A.). At last, protein bands were visualized using the enhanced chemiluminescence detection reagents (EMD Millipore) by a chemiluminescence imaging system (Tanon, Shanghai, China).

Yi R., Wang H., Deng C., Wang X., Yao L., Niu W., Fei M, & Zhaba W. (2020). Dihydroartemisinin initiates ferroptosis in glioblastoma through GPX4 inhibition. Bioscience Reports, 40(6), BSR20193314.

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Protein Expression Analysis by Western Blot

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Total protein was prepared using lysis buffer (0.1% triton X, TBS, phosphoblocker). After the separation of the cell lysate proteins by SDS‐PAGE electrophoresis, the cells were transferred to nitrocellulose membranes and blocked with 5% non‐fat milk. The membranes were then incubated overnight at 4°C with the following primary antibodies: Anti‐GPX4 antibody AB125066 (Abcam), anti‐NRF2 antibody #12721 (Cell Signaling Technology), and anti‐β‐actin (Sigma–Aldrich, St Louis). After incubation for 1 h at room temperature with peroxidase‐conjugated secondary antibody (Jackson Immuno Research Laboratories), the protein bands were examined using an ECL Western Blotting Detection Reagent and ImageQuant LAS 4000 mini software (GE Healthcare).

Watanabe E., Yokoi A., Yoshida K., Sugiyama M., Kitagawa M., Nishino K., Yamamoto E., Niimi K., Yamamoto Y, & Kajiyama H. (2022). Drug library screening identifies histone deacetylase inhibition as a novel therapeutic strategy for choriocarcinoma. Cancer Medicine, 12(4), 4543-4556.

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FLAG-Myc Protein Interaction Assay

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Target FLAG antibody in coating buffer (Roche) was coated onto ELISA plates (Nunc, Rochester, NY) at a 1:100 dilution. Coated FLAG antibody was then reacted with cell lysates from transfected HEK293A cells (prepared in RIPA buffer) for 1 h at room temperature and subsequently with anti-Myc antibody at a 1:500 dilution (4 °C, overnight). Then, the coated antibody was reacted with the peroxidase-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA). Finally, reaction buffer containing 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonate (Roche) was applied, and the absorbance was measured by a spectrometer at 405 nm with a reference at 490 nm.

Tamaki Y., Shodai A., Morimura T., Hikiami R., Minamiyama S., Ayaki T., Tooyama I., Furukawa Y., Takahashi R, & Urushitani M. (2018). Elimination of TDP-43 inclusions linked to amyotrophic lateral sclerosis by a misfolding-specific intrabody with dual proteolytic signals. Scientific Reports, 8, 6030.

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Quantifying Colonic Epithelial KLF4 Levels

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Colonic epithelial cells were isolated from the whole colon of rats and mice according to the method of Cherbuy et al. (1995) (link). The cell pellet from whole colon or HT29-MTX cell cultures was immediately used for protein extraction (Cherbuy et al., 2010 (link)) and Lowry’s procedure was used for protein assays. Western blot analysis was performed using 12% SDS-PAGE and anti-KLF4 (1:500; IMG-6081A, Imgenex, San Diego, CA, United States), with an appropriate peroxidase-conjugated secondary antibody (Jackson ImmunoResearch laboratories, West Grove, PA, United States). For each western blot, protein loads were determined using anti-cullin1 (1:200; sc-17775, Santa Cruz Biotechnology), or anti-GAPDH (1:1000; 905-734-100, Stressgen) antibodies. Signals detected on autoradiographic films were quantified by scanning densitometry using a Biovision 1000 and Bio-1D software (Vilber Lourmat, France).

Fernandez N., Wrzosek L., Radziwill-Bienkowska J.M., Ringot-Destrez B., Duviau M.P., Noordine M.L., Laroute V., Robert V., Cherbuy C., Daveran-Mingot M.L., Cocaign-Bousquet M., Léonard R., Robbe-Masselot C., Rul F., Ogier-Denis E., Thomas M, & Mercier-Bonin M. (2018). Characterization of Mucus-Related Properties of Streptococcus thermophilus: From Adhesion to Induction. Frontiers in Physiology, 9, 980.

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Immunohistochemical Staining and Quantification

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De-waxed sections were incubated in retrieval solution (pH 6.0), methanolic H₂O₂, and 0.5% Triton X-100, and subsequently washed in phosphate-buffered saline. Non-specific binding sites were blocked with 10% normal donkey serum (Jackson ImmunoResearch, West Grove, PA, USA). Sections were incubated with primary antibodies at 4 °C overnight and then with peroxidase-conjugated secondary antibody (Jackson ImmunoResearch) at room temperature (RT) for 2 h. Peroxidase activity was detected using 3,3′-diaminobenzidine (DAB; Vector Laboratories, Burlingame, CA, USA) as a chromogen. Stained tissues were viewed using an Olympus photomicroscope equipped with differential-interference contrast optics (BX 51; Olympus, Tokyo, Japan). Quantitative analysis was performed by calculating the percentage of positive area showing the same intensity using histogram equalization (TDI Scope Eye).

Lim S.W., Shin Y.J., Luo K., Quan Y., Cui S., Ko E.J., Chung B.H, & Yang C.W. (2019). Ginseng increases Klotho expression by FoxO3-mediated manganese superoxide dismutase in a mouse model of tacrolimus-induced renal injury. Aging (Albany NY), 11(15), 5548-5569.

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Western Blot Analysis of Osteogenic and Exosomal Markers

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Proteins in femurs, exosomes, or MC3T3-E1 cells were collected in RIPA buffer (Thermo Scientific, USA). The same amount of protein samples was loaded onto NuPage Bis–Tris polyacrylamide gels (Invitrogen, USA) and then proteins were transferred onto polyvinylidene difluoride membranes. After blocking in milk (5% w/v) for 4 h at room temperature, the membranes were subsequently incubated overnight at 4 °C with primary antibodies against GAPDH (1:5000; Proteintech, USA), Runx2 (1:1000; Cell Signaling Technology, USA), Bglap (1:500; Abcam, USA), Col1a1 (1:1000; Abcam, US), CD81 (1:1000; Abcam, USA), and TSG101 (1:1000; Abcam, USA). Next, the membranes were incubated with a peroxidase-conjugated secondary antibody (1:5000; Jackson, USA), and the signals were visualized using SuperSignal West substrate (Thermo Fisher Scientific, USA). The density was measured and analyzed using ImageJ software.

Wang Y., Zhang L., Wang K., Zhou H., Li G., Xu L., Hu Z., Cao X., Shi F, & Zhang S. (2022). Circulating Exosomes from Mice with LPS-Induced Bone Loss Inhibit Osteoblast Differentiation. Calcified Tissue International, 111(2), 185-195.

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