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Hrp linked goat anti rabbit antibody

Manufactured by Cell Signaling Technology
Sourced in United States

The HRP-linked goat anti-rabbit antibody is a secondary antibody used for the detection and visualization of primary rabbit antibodies in various immunoassays. It is conjugated with horseradish peroxidase (HRP), an enzyme that can catalyze a colorimetric reaction, enabling the detection of the target antigen.

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10 protocols using hrp linked goat anti rabbit antibody

1

Immunoblot Analysis of Plant Proteins

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Immunoblot analysis was performed as described previously (Fang et al., 2019) . For protein extraction, leaf samples were ground into powder in liquid nitrogen and homogenized in extraction buffer (100 mM HEPES, pH 7.5; 5 mM EDTA; 5 mM EGTA; 10 mM Na 3 VO 4 ; 10 mM NaF; 50 mM b-glycerophosphate; 1 mM phenylmethylsulphonyl fluoride; 10% [v/v] glycerol; 7.5% [w/v] polyvinylpolypyrrolidone; and 0.2% [v/v] b-mercaptoethanol), followed by centrifugation at 13,000 g for 20 min. After adjusting the concentration to the same level, proteins were then mixed with 53 loading buffer (125 mM Tris-HCl, pH 6.8; 5% [w/v] SDS; 25% [v/v] glycerol; 25% [v/v] b-mercaptoethanol; 3.13 mg bromophenol blue/5 mL buffer) and heated at 95°C for 10 min. SDSpolyacrylamide gel (12% [w/v]) electrophoresis (SDS-PAGE) was performed to separate the protein extracts.
For VDE and PsbS detection, antibodies specific to the VDE and PsbS proteins (Agrisera) were used, followed by incubation with a goat antirabbit HRP-linked antibody (Cell Signaling Technology 7074). For HY5 detection, antibodies specific to HY5 proteins (Shanghai Jiayuan Bio.) were used, followed by incubation with a goat antirabbit HRP-linked antibody (Cell Signaling Technology 7074). To test its mobility, a mouse monoclonal antibody recognizing HA (Pierce 26183) was used, followed by incubation with a goat antimouse IgG antibody (Millipore, AP124P).
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2

Investigating MAPK Signaling Pathways

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All reagents were purchased from Thermo Fisher Scientific if not specifically mentioned. RAW 264.7 treated with BBR3378 (100 ng/mL) concurrently with LPS challenge for 30 minutes or treated with BBR3378 at the same concentration but two hours prior to exposure of 30-minute LPS were lysed by using RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktail followed by centrifugation at 12,000 rpm for 15 min at 4°C to remove cell debris. For Western blot detection, samples were normalized for equal protein loading, boiled in sodium dodecyl sulfate (SDS) Laemmli sample buffer, resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membranes, and immunoblotted with antibodies specific for the proteins of interest followed by a horseradish peroxidase (HRP)-conjugated secondary antibody. Mitogen-activated protein kinase (MAPK) and phosphorylated MAPK (phospho-MAPK) family antibody sampler kits, β-Actin rabbit monoclonal antibody and HRP-linked goat anti-rabbit antibody were purchased from Cell Signaling Technologies (Danvers, MA). Chemiluminescent exposures were captured on a UVP ChemiDoc-It2 Imager (Thermo Fisher Scientific, Waltham, MA), and Immunoreactive bands were quantified using ImageJ (NIH Image, Bethesda, MD).
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3

Cinnamaldehyde Modulates Inflammatory Responses

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Cinnamaldehyde was purchased from Sigma-Aldrich (St.Louis, MO, USA). The total RNA Kit and Stool DNA Kit were purchased from OMEGA Bio-Tek (Norcross, GA, USA). One-step qRT-PCR kits were purchased from TOYOBO (Osaka, Japan). Anti-NF-κB p65, anti-phospho-NF-κB p65, anti-TNF, anti-IL-6 and HRP-linked goat anti-rabbit antibody were obtained from Cell Signaling Technology (Beverly, MA, USA). ECL Western Blotting Substrate was supplied by Thermo Fisher (Shanghai, China). RIPA lysis buffer and BCA protein assay kits were obtained from Beyotime (Shanghai, China). Nitrocellulose membranes were purchased from Sigma-Aldrich (St.Louis, MO, USA). AB/PAS staining kits were purchased from Solarbio Life Science (Beijing, China). Enzyme-linked immunosorbent assay (ELISA) kits for estimating TNF-α, and IL-6 levels were obtained from Multisciences Biotech (Hangzhou, China).
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4

Immunoblot Analysis of B Cell Proteins

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B220+ B cells were sorted from the spleen of AID+ ki/+ and WT mice, or Mb1+ ki/+ and WT mice. Then the RIPA extracts were fractionated on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels, electroblotted to polyvinylidene difluoride (PVDF) membranes and reacted with anti-c-MYC (Cell Signaling Technology, 9402), anti-BCL2 (Cell Signaling Technology, 3498) and anti-β-actin (Cell Signaling Technology, 4967) antibodies, and followed by HRP linked goat anti-rabbit antibody (Cell Signaling, 7074). HRP activity was determined using Immobilon Western Chemiluminescent reagent (Millipore, P90720).
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5

Striatal Protein Expression Analysis

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Striatum samples were homogenized by sonication in SDS buffer (10%). After determining the dynamic range of detection for each antibody, 20 μg of total protein were resolved in 4-20% SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes using the Trans-Blot Turbo transfer system (Bio-rad). Briefly, membranes were incubated overnight at 4°C with primary antibodies of interest anti-: TH (Cell Signaling; s58844), mTOR (Cell Signaling; s2983), p-mTOR (Cell Signaling; s5536), iNOS (Cell Signaling; s13120), MPC1 (Cell Signaling; s14462), pyruvate dehydrogenase (Cell Signaling; s3205), p-pyruvate dehydrogenase (Cell Signaling; s31866), acetyl-coenzyme A acetyltransferase 1 (Cell Signaling; s44276), carnitine palmitoyltransferase 1a (Cell Signaling; s97361), glutamate dehydrogenase (Cell Signaling; s12793), vinculin (Cell Signaling; s13901), βactin (Cell Signaling; s4970). After washing, membranes were incubated 1h at room temperature with HRP-linked goat anti-rabbit antibody (Cell Signaling; s7074). Bands were detected using enhanced chemiluminescence (ECL) and their densities quantified using ImageLab software (Bio-rad). Each band was delimited manually, and densities were normalized by dividing by the density of controls [38] (link).
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6

Striatal Protein Expression Analysis

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Striatum samples were homogenized by sonication in SDS buffer (10%). After determining the dynamic range of detection for each antibody, 20 μg of total protein were resolved in 4-20% SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes using the Trans-Blot Turbo transfer system (Bio-rad). Briefly, membranes were incubated overnight at 4°C with primary antibodies of interest anti-: TH (Cell Signaling; s58844), mTOR (Cell Signaling; s2983), p-mTOR (Cell Signaling; s5536), iNOS (Cell Signaling; s13120), MPC1 (Cell Signaling; s14462), pyruvate dehydrogenase (Cell Signaling; s3205), p-pyruvate dehydrogenase (Cell Signaling; s31866), acetyl-coenzyme A acetyltransferase 1 (Cell Signaling; s44276), carnitine palmitoyltransferase 1a (Cell Signaling; s97361), glutamate dehydrogenase (Cell Signaling; s12793), vinculin (Cell Signaling; s13901), βactin (Cell Signaling; s4970). After washing, membranes were incubated 1h at room temperature with HRP-linked goat anti-rabbit antibody (Cell Signaling; s7074). Bands were detected using enhanced chemiluminescence (ECL) and their densities quantified using ImageLab software (Bio-rad). Each band was delimited manually, and densities were normalized by dividing by the density of controls [38] (link).
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7

Immunoblotting Analysis of Pyroptosis Proteins

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Antibodies used for immunoblotting analysis were the following: anti-IL-1β (#12703), anti-caspase-1 (#3866), anti-caspase-4 (#4450), anti-caspase-5 (#46680), anti-MLKL (#14993), anti-phospho-MLKL (#91689), anti-RIP-3 (#13526), anti-phospho-RIP3 (#93654) and anti-Myc-tag (#2278) (Cell Signaling Technology); and anti-Flag (Sigma-Aldrich, F1804), anti-Bcl-2 (Santa Cruz Biotechnology, sc-783), anti-actin conjugated to horseradish peroxidase (Sigma-Aldrich, A3854), anti-GAPDH conjugated to horseradish peroxidase (Proteintech, HRP-60004), goat anti-rabbit HRP-linked antibody (#7074), and horse anti-mouse HRP-linked antibody (#7076) (Cell Signaling Technology). Anti-GSDMD was from (Novusbio, NBP2-33422). The LPS, nigericin and Z-VAD-FMK caspase inhibitor were purchased from Sigma-Aldrich. SYTOX Green nucleic acid stain was purchased from Invitrogen. Smac mimetic was from Tocris. Caspase-1, caspase-3 and Bcl-2 recombinant proteins were from EMD Millipore. Human TNFα was from R&D System. Smac mimic was from APExBIO.
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8

Immunoblot Analysis of Inflammasome Components

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Antibodies used for immunoblot analysis were the following: anti-NLRP3, anti-IL-1β, anti-caspase-1, anti-caspase-9 and anti-cytochrome c (Cell Signaling); and anti-Myc (Clontech Laboratories), anti-Flag (Sigma-Aldrich), anti-HA (Cell Signaling), anti-ASC (Santa Cruz Biotechnology), anti-actin conjugated to horseradish peroxidase (Sigma-Aldrich), goat anti-rabbit HRP-linked antibody, and horse anti-mouse HRP-linked antibody (Cell Signaling). Purified cytochrome c protein (Sigma-Aldrich), Profect-P1-lipid based protein delivery reagent (Targeting Systems), cardiolipin beads (Echelon), and the mitochondrial fractionation kit (Active Motif) were purchased and used as directed by the manufactures. The LPS, ATP, and Poly (dA-dT) were purchased from Sigma-Aldrich.
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9

Ischemic Cortex Western Blot Analysis

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Mice were euthanized and perfused with ice-cold saline. The ischemic cortex was sampled and homogenized using ultrasonication in RIPA buffer. Western blot analysis was performed on protein samples of equal mass quantified using a Bicinchoninic Acid Assay (BCA) assay.
The following primary antibodies were used: anti-β-actin (1:1000, 4970, Cell Signaling Technology, USA), anti-IL-1β (1:1000, 31202, Cell Signaling Technology, USA), anti-IL-6 (1:1000, 12912, Cell Signaling Technology, USA), anti-H3K4me3 (1:1000; cell-signaling technology, USA), anti-H3K4me1 (1:1000, 5326, Cell Signaling Technology, USA), and anti-H3 (1:1000, 4499, Cell Signaling Technology, USA). Goat anti-rabbit HRP-linked antibody (1:1000, 7074, Cell Signaling Technology, USA) served as the secondary antibody.
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10

Immunostaining and Western Blot Antibody Titrations

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Primary and secondary antibodies were used at the following concentrations: 1:1,600 mouse PECAM‐1 89C2 (Cell Signaling Technology), 1:100 rabbit RAB13 (Puro‐PLA, Millipore), 1:1,000 rabbit RAB13 (Western blotting and IF, Cambridge Bioscience), 1:3,500 mouse puromycin (Kerafast), 1:1,000 rabbit β‐tubulin 9F3 (Cell Signaling Technology), 1:200 mouse ZO‐1 1A12 (Thermo Fisher Scientific), 1:500 goat anti‐mouse Alexa Fluor 488 or Alexa Fluor 568 (Thermo Fisher Scientific), 1:500 goat anti‐rabbit Alexa Fluor 568 (Thermo Fisher Scientific), 1:5,000 goat anti‐mouse HRP‐linked (Cell Signaling Technology) and 1:5,000 goat anti‐rabbit HRP‐linked antibody (Cell Signaling Technology).
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