Goat anti rabbit hrp secondary antibody

Manufactured by Agilent Technologies

Sourced in Denmark, United States, Germany

The Goat anti-rabbit HRP secondary antibody is a laboratory reagent designed for use in immunoassays and other immunochemical techniques. It is a polyclonal antibody raised in goats against rabbit immunoglobulins and is conjugated to the enzyme horseradish peroxidase (HRP). The HRP label enables the detection and visualization of target antigens or proteins in various applications.

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Lab products found in correlation

Gelatin, by Reanal (2 mentions) Goat anti mouse hrp, by Agilent Technologies (1 mentions) Mowiol, by Polysciences (1 mentions) Iblot, by Thermo Fisher Scientific (1 mentions) Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions) Sonicator, by Qsonica (1 mentions) Epr18021, by Abcam (1 mentions) Clarity western ecl substrate, by Bio-Rad (1 mentions) Dc protein assay, by Bio-Rad (1 mentions) Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions) Gelcode blue safe protein stain, by Thermo Fisher Scientific (1 mentions) Pvdf membrane, by Thermo Fisher Scientific (1 mentions) A0080, by Agilent Technologies (1 mentions) Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Clarity max, by Bio-Rad (1 mentions) Sds page gel, by Bio-Rad (1 mentions) Laemmli buffer, by Bio-Rad (1 mentions) Chemidoc, by Bio-Rad (1 mentions) Hematoxylin, by Electron Microscopy Sciences (1 mentions) Nanozoomer slide scanner, by Hamamatsu Photonics (1 mentions)

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10 protocols using goat anti rabbit hrp secondary antibody

ELISA Quantification of Astrocyte Cytokines

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ELISA was performed as already described [99 (link)]. Briefly, (after titration in increasing dilutions) the astrocyte culture supernatants were diluted in a 1:1 ratio by ELISA coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) and were used for the coating of 96 well polystyrene ELISA plates (Maxisorp, NUNC Intermed, Copenhagen, Denmark). Free binding capacity of the wells was blocked by 1% gelatin (Reanal, Budapest, Hungary) in PBS. After washing with PBS, anti-IL-6 [98 (link)] and anti-TNFα [100 (link)] primary antibodies were added (2 h, 37 °C), followed by goat-anti-rabbit-HRP secondary antibodies (1:2000, DakoCytomation, Glostrup, Denmark). Colour reaction was developed by o-phenylene-diamine, and the optical density was measured at λ = 492 nm by ELISA plate reader (Titertek, Uniscan, Flow Laboratories, Helsinki, Finland).
Optical density data were averaged, and the standard error of mean values was calculated for each treatment. Bar charts showed relative changes in the expression level of cytokines in percentages, and the control level was set to 100%. Graphs show representative data out of three independent experiments. Statistically significant differences were calculated by one-way ANOVA followed by Student-Newman-Keuls pairwise comparison. The difference between groups was considered statistically different if p < 0.05.

Kozsurek M., Király K., Gyimesi K., Lukácsi E., Fekete C., Gereben B., Mohácsik P., Helyes Z., Bölcskei K., Tékus V., Pap K., Szűcs E., Benyhe S., Imre T., Szabó P., Gajtkó A., Holló K, & Puskár Z. (2023). Unique, Specific CART Receptor-Independent Regulatory Mechanism of CART(55-102) Peptide in Spinal Nociceptive Transmission and Its Relation to Dipeptidyl-Peptidase 4 (DDP4). International Journal of Molecular Sciences, 24(2), 918.

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EGFR Signaling Pathway Analysis

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Streptavidin-coated quantum
dots of 565 nm
and biotinylated EGF were from Life Technologies. Mouse anti-phospho
p42/44 MAPK (clone MAPK-YT) antibody was from Sigma. Mouse anti-phospho
p42/44-ERK1/2 and rabbit anti total-ERK1/2 antibodies were from Cell
Signaling Technologies. Mouse monoclonal anti-EGFR antibody clone
F4 binding to the sequence DVVDADADEYLIPQ, which corresponds to EGFR
amino acid residues 985–996, was obtained from the monoclonal
antibody facility at Cancer Research UK. Donkey anti-mouse secondary
antibodies conjugated to either DyLight594 or Cy2 were from Jackson
Immuno Research, and goat anti-mouse-HRP and goat anti-rabbit-HRP
secondary antibodies were from Dako. Unconjugated EGF was from Peprotech.
Sphero polystyrene beads (average diameter 1.23 μm) was from
Spherotech and Mowiol from Polysciences. All standard chemicals were
from Sigma-Aldrich or VWR.

Zhang R., Fruhwirth G.O., Coban O., Barrett J.E., Burgoyne T., Lee S.H., Simonson P.D., Baday M., Kholodenko B.N., Futter C.E., Ng T, & Selvin P.R. (2016). Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-resolution Microscopy. ACS Nano, 11(1), 249-257.

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Drosophila Hemolymph Protein Analysis

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For Western blots, hemolymph samples were collected from 25 flies in a protease inhibitor solution as described above. Protein concentration of the samples was determined by Bradford assay. 25 µg of protein extract was separated on a 4–20% acrylamide precast Novex gel (Invitrogen) under reducing conditions and transferred to nitrocellulose membranes (Invitrogen iBlot). After blocking in 2% bovine serum albumin in PBT for 1 h, samples were incubated at 4°C overnight with rabbit antibodies against Drosophila PPO1 or PPO2 [13] (link) in a 1∶2000 dilution. Goat anti-rabbit-HRP secondary antibody (Dako) in a 1∶2000 dilution was incubated for 2 h at room temperature. Bound antibody was detected using ECL (GE Healthcare) according to the manufacturer's instructions.

Binggeli O., Neyen C., Poidevin M, & Lemaitre B. (2014). Prophenoloxidase Activation Is Required for Survival to Microbial Infections in Drosophila. PLoS Pathogens, 10(5), e1004067.

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ELISA Quantification of Cytokine Levels

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ELISA was performed as already described (Holló et al., 2000 (link)). Briefly, (after titration in increasing dilutions) the astrocyte culture supernatants were diluted in 1:1 ratio by ELISA coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) and used for coating of 96 well polystyrol ELISA plates (Maxisorp, NUNC Intermed, Copenhagen, Denmark). Free binding capacity of the wells was blocked by 1% gelatin (Reanal, Budapest, Hungary) in PBS. After washing with PBS anti-IL-6, anti-GM-CSF or anti-CCL-5 primary antibodies were added (1:1000, 2 h, 37°C), followed by goat-anti-rabbit-HRP secondary antibody (1:500, DAKO). Color reaction was developed by o-phenylenediamine and the optical density was measured at λ = 492 nm by ELISA plate reader (Titertek, Uniscan). Graphs represent results of three independent experiments.

Gajtkó A., Bakk E., Hegedűs K., Ducza L, & Holló K. (2020). IL-1β Induced Cytokine Expression by Spinal Astrocytes Can Play a Role in the Maintenance of Chronic Inflammatory Pain. Frontiers in Physiology, 11, 543331.

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Western Blotting of p53 and p21 in B16-F10 Cells

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Whole lysates from B16-F10 p53^+/+ and p53^−/− cells were probed for p53 and p21 expression. Cells were treated for 24 hours with 1.5 μmol/L Navtemadlin (to activate p53) or with DMSO control, lysed using 1 × Laemmli lysis buffer, heated at 95°C for 5 minutes, and sonicated (Qsonica Sonicators) for 30 seconds at 20% amplitude. After protein concentrations were determined using the DC Protein Assay (Bio-Rad), lysates were loaded (18 μg/lane) in a 4%–15% polyacrylamide gel (Mini-Protein TGX Stain-Free 12 well, Bio-Rad), and electrophoresed for 45 minutes at 150 V. Proteins were then transferred using a semidry method onto polyvinylidene difluoride membranes for 30 minutes according to manufacturer's instructions (Bio-Rad). Once membranes were blocked for 1 hour with blocking buffer (5% milk made in PBS containing 0.1% Tween-20), they were incubated overnight at 4°C with rabbit anti-mouse p53 (Abcam, EPR20416-124, 1:1,000) or rabbit anti-mouse p21 (Abcam, EPR18021, 1:1,000) primary antibodies made in blocking buffer. Membranes were subsequently washed in PBS containing 0.1% Tween-20, incubated for 1 hour at room temperature with goat anti-rabbit HRP secondary antibody (Dako, 1:1,000), and washed again. Protein expression was detected using chemiluminescence (Clarity Western ECL Substrate, Bio-Rad).

Ingelshed K., Spiegelberg D., Kannan P., Påvénius L., Hacheney J., Jiang L., Eisinger S., Lianoudaki D., Lama D., Castillo F., Bosdotter C., Kretzschmar W.W., Al-Radi O., Fritz N., Villablanca E.J., Karlsson M.C., Wermeling F., Nestor M., Lane D.P, & Sedimbi S.K. (2022). The MDM2 Inhibitor Navtemadlin Arrests Mouse Melanoma Growth In Vivo and Potentiates Radiotherapy. Cancer Research Communications, 2(9), 1075-1088.

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Fibrin Clot Protein Analysis

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Clots were made using plasma by the addition of thrombin (0.5 U/ml) and CaCl₂ (5 mM), with and without FXIII inhibitor T101 (1 mM). The fibrin film was removed from each clot and reduced by the addition of NuPAGE sample reducing agent (100 mM DTT) and heating at 95°C for 15 minutes. Fibrin samples were prepared by the formation of a clot with IF-1 fibrinogen (1 mg/ml), addition of thrombin and CaCl₂, and reduction as described above. FXIII-, BSA-, and IF-1–purified fibrinogen samples were reduced in a similar way and run alongside the films to help identify bands in the gel, and as controls for the blots. Protein concentrations were determined using NanoDrop to load 2 μg of each protein sample on 2 identical 4%–12% NuPAGE Bis-Tris gels. After running, one gel was stained using GelCode Blue Safe Protein Stain (Thermo Fisher Scientific), and one was transferred to a PVDF membrane (Thermo Fisher Scientific). The membrane was blocked overnight using 4% skim milk in 50 mM Tris, 150 mM NaCl, 0.1% Tween-20. Polyclonal rabbit anti–human fibrinogen antibody (A0080; Dako) was added to the blot in blocking buffer and detected using goat anti-rabbit HRP secondary antibody (P0448; Dako). Signal was detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).

Macrae F.L., Duval C., Papareddy P., Baker S.R., Yuldasheva N., Kearney K.J., McPherson H.R., Asquith N., Konings J., Casini A., Degen J.L., Connell S.D., Philippou H., Wolberg A.S., Herwald H, & Ariëns R.A. (2018). A fibrin biofilm covers blood clots and protects from microbial invasion. The Journal of Clinical Investigation, 128(8), 3356-3368.

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Western Blotting for Pericyte Signaling

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For western blotting, pericytes were seeded in 24-well plates (30,000 cells/well). The cells were lysed directly in the well using 100 μl of 1× Laemmli buffer (Bio-Rad) supplemented with 0.1 M DTT. The lysates were denatured at 95°C for 5 min and run on 15-well 4-15% SDS-PAGE gels (Bio-Rad) before being blotted onto Turbo-transfer-packs (Bio-Rad). The post-transfer nitrocellulose membranes were then blocked for 1 h in 5% milk in Tris-buffered saline with 0.1% Tween-20 (TBST). Next, the membranes were incubated with the primary antibodies (Table S1) in 5% BSA in TBST O/N. The membranes were subsequently washed 3× with TBST before goat-anti-rabbit-HRP secondary antibody was added (1:5000, Dako) in 5% milk in TBST and incubated at RT for 1 h. Membranes were then washed 3× with TBST followed by protein detection using HRP substrates Clarity or Clarity Max (Bio-Rad) to measure the chemiluminescence on a ChemiDoc (Bio-Rad).
For evaluating signalling mechanisms of PDGFRβ and intracellular phosphorylation of MAPK targets, siRGS5 or siCTRL pericytes were exposed to 8 h of hypoxia in deoxygenated (0.5-0.7% O₂) serum-free DMEM/F12 medium supplemented with 15 nM HEPES before being stimulated with either S1P (1 μM) or PDGFBB (5 ng/ml) for 5 min, directly lysed and analysed by western blotting. For additional information on antibody validation, see Table S3.

Enström A., Carlsson R., Özen I, & Paul G. (2022). RGS5: a novel role as a hypoxia-responsive protein that suppresses chemokinetic and chemotactic migration in brain pericytes. Biology Open, 11(10), bio059371.

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Histological Analysis of Fracture Healing

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Following sacrifice, injured limbs from stress fracture (n = 22) and full fracture (n = 15) were harvested and placed in 10% neutral buffered formalin for 24 hrs. Tissue was decalcified for 14 days using 14% EDTA, embedded in paraffin, and longitudinal cuts were made for histological observation and immunohistochemistry (IHC) processing. Histological sections of each time point were stained with hematoxylin and eosin (H&E) to show the progression of healing in each fracture type at each time point in the study (Figure 2).
Antibodies for Gr-1 (Bio-Rad MCA2387T, 1:500 dilution), F4/80 (Bio-Rad, MCA497RT 1:500 dilution), and CD45 (BD Pharmingen 550539, 1:500 dilution) were used with an ImmPRESS Reagent Anti-Rat IgG (VectaShield MP-7404) kit to stain for infiltration of neutrophils, macrophages, and leukocytes, respectively. An antibody for p-Akt T308 (Cell Signaling #2965) was used with a goat anti-rabbit/HRP secondary antibody (Dako P0448) in order to stain for activation of the PI3K-Akt signaling pathway. Sections were counterstained with hematoxylin (Modified Mayer’s, Electron Microscopy Services). Following staining, slides were imaged using a Nanozoomer slide scanner (Hamamatsu) at 20X magnification.

Coates B.A., McKenzie J.A., Buettmann E.G., Liu X., Gontarz P.M., Zhang B, & Silva M.J. (2019). Transcriptional Profiling of Intramembranous and Endochondral Ossification after Fracture in Mice. Bone, 127, 577-591.

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Immunohistochemical Analysis of Chronic MS Lesions

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Paraffin-embedded brain samples of chronic active lesions from four MS patients were provided by the UK Multiple Sclerosis Tissue Bank and stained with hematoxylin and eosin (HE) and Klüver-Barrera (KB) for inflammation and demyelination assessment. Four-micrometer-thick, paraffin-embedded serial sections were deparaffined in xylene and rehydrated in alcohol. Endogenous peroxidase activity was blocked with hydrogen peroxide (2%), methanol (70%), and PBS for 20 min. Antigen retrieval was performed in TE buffer (1 M TrismaBase and 1 mM EDTA) (pH = 9) in the microwave. Non-specific protein binding was blocked with 0.2% of bovine albumin (BSA) in PBS. Sections were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-CPXM2 (Biorbyt), rabbit anti-NLRP9 (Abcam), and rabbit anti-IGSG9B (Abcam). Samples were incubated for 1 h at room temperature with goat-anti rabbit HRP secondary antibody (Dakocytomation) and stainings were visualized with 3,3′diaminobenzidine (Sigma, St Louis, MO, USA) as a chromogenic substrate.

Gil-Varea E., Urcelay E., Vilariño-Güell C., Costa C., Midaglia L., Matesanz F., Rodríguez-Antigüedad A., Oksenberg J., Espino-Paisan L., Dessa Sadovnick A., Saiz A., Villar L.M., García-Merino J.A., Ramió-Torrentà L., Triviño J.C., Quintana E., Robles R., Sánchez-López A., Arroyo R., Alvarez-Cermeño J.C., Vidal-Jordana A., Malhotra S., Fissolo N., Montalban X, & Comabella M. (2018). Exome sequencing study in patients with multiple sclerosis reveals variants associated with disease course. Journal of Neuroinflammation, 15, 265.

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Western Blot Analysis of RAG-2 in Retinal Tissues

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Individual adult tissues and embryonic retinas were processed as previously described^{19 (link)}. Briefly, tissues or retinas were lysed, and 50 μg of total protein was resolved by PAGE and transferred to PVDF membranes. Membranes were blocked and incubated with antibodies against RAG-2 (1/1000, Abcam #ab133609; Cambridge, UK), and subsequently with a polyclonal goat anti-rabbit HRP secondary antibody (Dako Cytomation, Glostrup, Germany).

Álvarez-Lindo N., Baleriola J., de los Ríos V., Suárez T, & de la Rosa E.J. (2019). RAG-2 deficiency results in fewer phosphorylated histone H2AX foci, but increased retinal ganglion cell death and altered axonal growth. Scientific Reports, 9, 18486.

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