ELISA for PE binding was performed by overnight coating of high binding 96-well plates (Corning Incorporated) with 50 μl of 4 μg/ml PE in PBS. Plates were then washed with PBS containing 0.05% TWEEN 20 (Sigma) and then blocked with PBS containing 1 % PBS. Monoclonal antibodies were incubated for 1 hour at indicated dilutions in PBS. Plates were then washed and incubated with HRP conjugated anti-mouse IgG (Jackson Immunoresearch). Polyreactivity was assessed using LPS from E. coli (Sigma), human insulin (Sigma), dsDNA (Sigma) and ssDNA (prepared from dsDNA by heating at 95° C for 30 minutes), and KLH (Sigma) (Mouquet et al., 2010 (link)). High binding plates were incubated overnight with 50 μl of ssDNA, dsDNA, LPS, and KLH at 10 μg/ml each and insulin at 5 μg/ml. Plates were then washed with PBS containing 0.001% TWEEN 20. Plates were blocked for 2 hours at room temperature in PBS containing 1 mM EDTA, 0.05% TWEEN 20 and 2.5% bovine serum albumin (Sigma). After washing, monoclonal antibodies were incubated for 2 hours at indicated dilutions in PBS. Plates were then washed and incubated with HRP conjugated anti-mouse IgG (Jackson Immunoresearch) for 1 hour. After washing, plates were developed by adding HRP substrate (Life Technologies). Absorbance was measured at 405 nm.
Hrp conjugated anti mouse igg
Manufactured by Jackson ImmunoResearch
Sourced in United States
HRP-conjugated anti-mouse IgG is a secondary antibody reagent that binds to mouse immunoglobulin G (IgG) molecules. The antibody is conjugated to horseradish peroxidase (HRP), an enzyme that can be used for signal detection in various immunoassays and immunochemical techniques.
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Lab products found in correlation
Hrp conjugated anti rabbit igg, by Jackson ImmunoResearch (11 mentions)
Immobilon p, by Merck Group (4 mentions)
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Casein blocking buffer, by Merck Group (3 mentions)
Horseradish peroxidase hrp conjugated anti rabbit igg, by Jackson ImmunoResearch (3 mentions)
Tau2 18 peptide, by GenScript (3 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (3 mentions)
Anti flag, by Merck Group (3 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
P iκbα, by Cell Signaling Technology (2 mentions)
Ecl prime, by GE Healthcare (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Hrp conjugated anti goat igg, by Jackson ImmunoResearch (2 mentions)
Emax microplate reader, by Molecular Devices (2 mentions)
Supersignal, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Supersep ace, by Fujifilm (2 mentions)
Signal enhancer hikari, by Nacalai Tesque (2 mentions)
Mouse anti α tubulin, by Merck Group (2 mentions)
70 protocols using hrp conjugated anti mouse igg
1
ELISA for Polyreactive Antibody Binding
2
Immunohistochemical Analysis of Neural and Proliferative Markers
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After the 7 μm-thick sections were deparaffinized and rehydrated, endogenous peroxidase activity was inhibited by treatment with 3% H2O2 in methanol for 15 min. The sections were rinsed in Tris-HCl phosphate buffer (10 mM Tris, 120 mM NaCl, 8.4 mM Na2HPO4 and 3.5 mM KH2PO4; pH 7.8) and then incubated overnight at room temperature with the following primary antibodies diluted in 1% bovine serum albumin (BSA) in Tris-HCl phosphate buffer: anti-βIII-tubulin (1:1000, Promega, Madison, WI, USA), anti-PCNA (1:400, DAKO, Carpinteria, CA, USA) and anti-4-bromo-2′-deoxyuridine (BrdU; 1:200 Roche, Penzberg, Germany). The sections were rinsed and subsequently incubated for 2 h at room temperature with HRP-conjugated mouse anti-IgG (Jackson ImmunoResearch, West Grove, PA, USA). The peroxidase activity was developed using diaminobenzidine and H2O2. For immunohistochemical analysis of BrdU, the sections were treated with 2N HCl for 30 min at 37 °C and incubated with 5% BSA for 30 min before being incubated with the primary antibody. The images were obtained using an Axioplan 2 microscope (Carl Zeiss, Oberkochen, Germany) connected to a digital camera (Nikon, Digital Camera DXM1200, Melville, NY, USA). In all cases, omission of the primary antibody served as the negative control (Figure S1 ).
3
Immunohistochemical Analysis of Cellular Markers
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After the 7-lm-thick sections were deparaffinized and rehydrated, endogenous peroxidase activity was inhibited by treatment with 3% H 2 O 2 in methanol for 15 minutes. The sections were rinsed in Tris-HCl phosphate buffer (10 mM Tris, 120 mM NaCl, 8.4 mM Na2HPO4, and 3.5 mM KH2PO4, pH 7.8) and then incubated overnight at room temperature with the following primary antibodies, which were diluted in 1% bovine serum albumin (BSA) in Tris-HCl phosphate buffer: anti-vimentin (1:200, DAKO, Carpinteria, CA, USA, http://www. dako.com/); anti-bIII tubulin (1:1000, Promega, Madison, WI, USA, http://www.promega.com/); and anti-BrdU (1:200, Roche Diagnostics, Risch-Rotkreuz, Switzerland, http://www.rochediagnostics.com/). The sections were rinsed and subsequently incubated for 2 hours at room temperature with the appropriate secondary antibody conjugated to HRP, HRP-conjugated mouse anti-IgG (Jackson ImmunoResearch, West Grove, PA, USA, https://www.jacksonimmuno.com/). The peroxidase activity was developed using diaminobenzidine and H 2 O 2 [19] . For immunohistochemical analysis of BrdU, the sections were www. StemCells ). In all cases, omission of the primary antibody served as the negative control [20] .
4
Molecular Profiling of Melanogenesis
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Anti-MITF (C5), anti-EDNRB (EPR7013), anti-CREB (48H2), anti-phospho-CREB (87G3), anti-β-actin (AC-15), anti-rabbit IgG HRP-conjugated and H89 dihydrochloride were purchased from Abcam (Cambridge, MA). Anti-mouse IgG HRP-conjugated was purchased from Jackson ImmunoResearch (West Grove, PA). Antibodies for MAPK and phosphorylated MAPK, the MAPK family sampler kit and the phospho-MAPK family sampler kit were purchased from Cell Signaling Technology (Beverly, MA). Antibodies for MSK1 (C27B2) and phosphorylated (S376 and T581) MSK1 were purchased from Cell Signaling Technology. For Real-time RT-PCR, primers for β-actin (Hs_ACTB_1_SG Quantitect Primer Assay; QT00095431), EDNRB (Hs_EDNRB_1_SG Quantitect Primer Assay; QT00014343) and MITF (Hs_MITF_1_SG Quantitect Primer Assay; QT00037737) were purchased from Qiagen (Hilden, Germany). PBE (Flavangenol) which obtained by hot water extraction method from French maritime pine (Pinus pinaster) bark was supplied by Toyo Shinyaku (Saga, Japan).
5
Western Blot Analysis of Protein Samples
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Defined amounts of protein per sample was diluted in 4 × SDS-PAGE sample buffer, denatured for 10 min at 95 °C, and loaded on a 10% or 15% acrylamide gel. Transfer to a nitrocellulose membrane (Amersham) was performed in a semi-dry blotting instrument, applying 1 mA/cm2 of membrane surface for 90 min. The membrane was incubated for 1 h in blocking solution (PBS-T with 5% w/v powder milk). All antibodies were also diluted in blocking solution (anti-AIP/Alix, 1:500 [ABC40, Sigma- Aldrich], anti-Flotillin2, 1:400 [610383, BD Biosciences], anti-FRα, 1:2000 [NCL-L-FRalpha, Leica Biosystems], anti-PTPS, 1:2000 [PA5-22121, Thermo Fisher Scientific], anti-TOM20, 1:1000 [sc-11415, Santa Cruz Biotechnology], anti-mouse IgG HRP-conjugated, 1:10,000 [Jackson ImmunoResearch Laboratories], anti-rabbit IgG HRP-conjugated, 1:5000 [Jackson ImmunoResearch Laboratories]). Membranes were incubated in primary antibody overnight at 4 °C, followed by 3 5-min washes in PBS-T and 1 h incubation in secondary antibody solution RT. Lumi-LightPLUS Western Blotting Substrate (Sigma-Aldrich) was used to visualize the peroxidase-conjugated secondary antibody in the Luminescent image reader LAS-400 mini (Fujifilm). WB images were analyzed using ImageJ (National Institutes of Health).
6
Zika Virus Envelope Protein Analysis
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The purified ZIKV_E recombinant proteins were resolved by 15% SDS-PAGE under reducing conditions with beta-mercaptoethanol (β-me). For western blot analysis, rabbit anti-mouse IgG HRP-conjugated (10,000× dilution) (Jackson ImmunoResearch, West Grove, PA, USA) detected ZIKV_E recombinant proteins, and the blot was developed using the 3,3′-diaminobenzedine (DAB)/H2O2 system.
7
SARS-CoV-2 Spike Protein ELISA Assay
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ELISA assays were performed in 96-microwell plates coated with 150 ng/well of SARS-CoV-2 Spike (aa 16–1213) His-tagged recombinant protein (ThermoFisher) and 1010 phage or AAVP particles/50 μL of PBS O.N. at 4°C (Nunc MaxiSorp flat bottom, ThermoFisher Scientific). Coated plates were blocked with PBS containing 5% low-fat milk and 1% BSA for 1 h at 37°C. Two-fold serial dilutions (starting at 1:32) or 1:50 fixed dilution of sera in blocking buffer were added to separate the wells and incubated for 1–2 h at 37°C. Following three washes with PBS and PBS containing 0.1% of Tween 20, bound antibodies were detected with an anti-mouse IgG HRP-conjugated (Jackson ImmunoResearch) at optical density (OD) at 450 nm. Commercially available polyclonal IgG anti-Spike protein antibody (Thermo Fisher, MA5–35949) or anti-fd bacteriophage antibody (Sigma Aldrich) served as positive controls.
8
Modulation of Inflammation Pathways by Medicinal Plant Extracts
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Cells were pre-treated with EtOAc fractions of P. nudicaule or P. rhoeas for 1 h and then incubated with LPS (5 ng/mL) for 30 min–24 h. Nuclear, cytoplasmic, and whole-cell extracts were prepared, and western blotting was performed as described previously [29 (link)]. Primary antibodies against Nrf2, lamin B, cyclooxygenase (COX) 2, inducible NO synthase (iNOS), p65, phospho (p)-p65, IκBα, p-IκBα, STAT3, and p-STAT3 were purchased from Cell Signaling Technology (Danvers, MA, USA). β-actin was from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). HRP-conjugated anti-mouse IgG and anti-rabbit secondary antibodies (1:5000–1:10,000) were from Jackson Immuno Research Laboratories, Inc. (West Grove, PA, USA). ImageJ software (https://imagej.nih.gov/ij/ , accessed on 5 November 2021). was used for densitometric analysis of each protein band. Data are presented as mean ± Standard Deviation (SD) from at least three independent experiments.
9
SARS-CoV-2 Spike Protein RBD ELISA
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The serum collected at each time point was evaluated for binding titers. In this assay, 96-well ELISA plates (NUNC) were coated with 1 μg/mL recombinant SARS-CoV-2 spike RBD-His protein (Sino Biological, 40592-V08B, Beijing, China) in PBS, overnight at 4 °C. The plates were washed three times with 0.05% PBST (Tween 20 in PBS) and blocked with 5% skim milk in 0.05% PBST (SM) for 2–3 h at room temperature. The sera were serially diluted in 5% SM, added to the wells, and incubated for 2 h at 37 °C. Following incubation, the plates were washed five times with 0.05% PBST and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, 115-035-003, West Grove, PA, USA), IgG1 (Jackson ImmunoResearch Laboratories 115-035-205), or IgG2a (Jackson ImmunoResearch Laboratories 115-035-206) for 1 h at 37 °C. After the final wash, the plates were developed using TMB solution (Surmodics, TMBW-1000-01, Eden Prairie, MN, USA), and the reaction was stopped with 2N H2SO4. The plates were analyzed at 450 nm using a SpectraMax Plus384 (Molecular Devices, San Jose, CA, USA).
10
Western Blotting of Cellular Proteins
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Western blotting was processed as described previously14 (link). Briefly, MDBK cells were lysed and the cellular proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was probed with one of the following primary antibodies, mouse anti-HO-1 polyclonal antibodies at a 1:1000 dilution and mouse anti-NS5B of BVDV polyclonal antibodies at a 1:1000 dilution produced in our laboratory, anti-HO-2 antibody at a 1: 1000 (Abcam, catalog#: ab90492, Cambridge, UK), anti-α-tubulin antibody at a 1:5000 (Abcam, catalog#: ab7750, Cambridge, UK), and then with HRP-conjugated anti-mouse IgG at a 1:2000 (Jackson, catalog#: 111-035-003, West Grove, PA, USA) as the secondary antibody. Immunolabeled proteins were visualized using ECL reagent (Pierce, Rockford IL, CA, and USA).
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