Horseradish peroxidase hrp conjugated secondary antibodies

Manufactured by Dianova

Sourced in Germany

Horseradish peroxidase (HRP)-conjugated secondary antibodies are a type of laboratory reagent used in various immunoassays and detection methods. HRP is an enzyme that catalyzes the oxidation of substrates, producing a colored or luminescent signal that can be detected and quantified. These secondary antibodies are conjugated with HRP, allowing for the indirect detection of target proteins or molecules in samples.

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Lab products found in correlation

Phosstop, by Roche (1 mentions) Hybond p, by GE Healthcare (1 mentions) Chemocam imager, by Intas (1 mentions) Hybond, by Cytiva (1 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions) Bca assay, by Thermo Fisher Scientific (1 mentions) Polyvinylidene fluoride membrane, by Merck Group (1 mentions) Versadoc mp 400 system, by Bio-Rad (1 mentions) Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Ab6671, by Abcam (1 mentions) Alexa 488, by Thermo Fisher Scientific (1 mentions) Cyanine3 (cy3), by Dianova (1 mentions)

Close spelling variants of this product

Horseradish peroxidase (14 citations) Peroxidase-conjugated secondary antibodies (4 citations)

4 protocols using horseradish peroxidase hrp conjugated secondary antibodies

Protein Extraction and Western Blot Analysis

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Tissue was lysed in sucrose buffer containing 320 mM sucrose, 10mM Tris-HCL (pH 7.4), 1mM NaHCO
₃, 1mM MgCl
₂, 1% Triton X-100, 2% lithiumdodecylsulfate, 0.5% sodiumdeoxycholate, and protease and phosphatase inhibitors (cOmplete™, PhosSTOP™, Roche). 25 µg (brain tissue) and 20 µg (lung, liver, kidney, small intestine, heart) of protein lysates were resolved on 12% SDS-polyacrylamide gels under denaturing conditions and electro-transferred to PVDF membranes (Hybond P; GE Healthcare). Blocking was performed for 1h in Tris buffered saline / 0.05% Tween 20 (TBST) containing 5% milk powder and incubated in primary antibody at 4°C overnight in the same solution. Membranes were washed in TBST prior to incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000 Dianova, Hamburg) for 1h. Blots were developed by enhanced chemiluminescence (Pierce, Rockford) and scanned using the ChemoCam Imager (Intas Science Imaging Instruments, Goettingen).

Winchenbach J., Düking T., Berghoff S.A., Stumpf S.K., Hülsmann S., Nave K.A, & Saher G. (2016). Inducible targeting of CNS astrocytes in Aldh1l1-CreERT2 BAC transgenic mice. F1000Research, 5, 2934.

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Western Blot Analysis of pERK/ERK in Mice

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Frozen tissue samples were lysed in RIPA lysis buffer on ice, and protein was isolated. Protein quantification was done by BCA assay (Thermo Scientific, USA). Fifty micrograms of protein was loaded on a 10% polyacrylamide gel for electrophoresis. Proteins were blotted to polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). After blocking in 3% bovine serum albumin (BSA), incubation with primary antibodies was done overnight at 4°C. The following antibodies were used: pERK (44/42 kDa, 9102S), ERK1/2 (44/42 kDa, 9106S, both from Cell Signaling Technologies, USA). 14-3-3 served as loading control (28 kDa, K-19, Santa Cruz Biotechnology, USA). After washing in PBS horseradish peroxidase (HRP) conjugated secondary antibodies (Dianova, Hamburg, Germany) were incubated for 1 h at room temperature. Proteins were visualized by incubation with SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, USA). To enhance signal intensity SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, USA) was used according to the manufacturer's instructions. Bands were immediately captured by VersaDoc MP 400 System (Bio Rad, USA). Five mice per group were analyzed.

Wang L., Vijayan V., Jang M.S., Thorenz A., Greite R., Rong S., Chen R., Shushakova N., Tudorache I., Derlin K., Pradhan P., Madyaningrana K., Madrahimov N., Bräsen J.H., Lichtinghagen R., van Kooten C., Huber-Lang M., Haller H., Immenschuh S, & Gueler F. (2019). Labile Heme Aggravates Renal Inflammation and Complement Activation After Ischemia Reperfusion Injury. Frontiers in Immunology, 10, 2975.

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Western Blot Analysis of Sciatic Nerve

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Sciatic nerves (n = 5 per group) were lysed by ultrasonication in 60 mM Tris-HCl, pH 6.8, containing 2% sodium dodecyl sulfate (SDS) and 10% sucrose. Tissue lysates were diluted 1:1 in sample buffer (250 mM Tris-HCl, pH 6.8, containing 4% SDS, 10% glycerol, and 2% b-mercaptoethanol) and denatured at 95°C for 5 min. Protein concentration was assessed with the bicinchoninic acid assay (BCA assay) protein assay (Pierbo Science, Bonn, Germany). Proteins (20 µg per lane) were separated by electrophoresis on a 12.5% or 15% SDS-polyacrylamide gel and transferred to nitrocellulose by electroblotting. Nonspecific binding sites were blocked with 5% dried milk powder for 45 min. The blots were incubated with rabbit polyclonal anti-TNFα (ab 6671; 1:2000; Abcam, MA, USA) at 4°C overnight. Proteins were detected by incubating with horseradish peroxidase (HRP)-conjugated secondary antibodies at a 1:4,000 dilution; Dianova) at RT for 2 h and chemiluminescence kit (Amersham, Pharmacia, Freiburg, Germany). Integrated optical densities of the immunoreactive protein bands were measured with Gel Analyzer software (Media Cybernetics, Silver Spring, MD). Equal protein loading was verified using mouse anti-D-glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH, Research Diagnostics, Flanders, The Netherlands; 1:3000).

Kosacka J., Woidt K., Toyka K.V., Paeschke S., Klöting N., Bechmann I., Blüher M., Thiery J., Ossmann S., Baum P, & Nowicki M. (2019). The role of dietary non-heme iron load and peripheral nerve inflammation in the development of peripheral neuropathy (PN) in obese non-diabetic leptin-deficient ob/ob mice. Neurological research, 41(4).

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Immunoblotting and Immunofluorescence Protocols

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The antibodies used in immunoblotting analyses of polypeptides separated by SDS-PAGE, immunofluorescence, and immunoelectron microscopy are listed in Tables 1 and 2 and in previous publications (e.g., Franke et al. 2013 Franke et al. , 2015;; Rickelt et al. 2011; Domke et al. 2014) .
Primary antibodies were visualized with goat anti-mouse, goat anti-guinea pig, goat anti-rat, or goat anti-rabbit IgG (H+L) secondary antibodies conjugated to Cy3 (Dianova, Hamburg, Germany) or Alexa 488 (Invitrogen, Karlsruhe, Germany). Immunoblot analysis was performed with horseradish-peroxidase (HRP)-conjugated secondary antibodies (Dianova).

Domke L.M, & Franke W.W. (2019). The cell-cell junctions of mammalian testes: II. The lamellar smooth muscle monolayer cells of the peritubular wall are laterally connected by vertical adherens junctions-a novel architectonic cell-cell junction system. Cell and tissue research, 375(2).

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