Marc-145 cells or PAMs were infected with PRRSV. At 48 h post-infection, the cells were treated with RIPA lysis buffer, and the samples were separated with SDS–PAGE (15%). Then, the proteins were transferred to PVDF membranes. Western blotting was performed as previously reported [19 (link)]. An anti-N protein antibody (SOW17, Median, Seoul, Republic of Korea) (1:1000 dilution) and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Beyotime, Nantong, China) were used as primary and secondary antibodies, respectively. Finally, the membranes were analyzed using Biosystems C280 (Azure, Dublin, CA, USA).
Horseradish peroxidase hrp conjugated goat anti mouse igg
Manufactured by Beyotime
Sourced in China
Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG is a secondary antibody used in immunoassays and immunohistochemistry. It consists of goat-derived antibodies specific to mouse IgG that are covalently linked to the enzyme horseradish peroxidase.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (1 mentions)
Radio immunoprecipitation assay ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Anti gapdh mouse monoclonal antibody, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Thermo Fisher Scientific (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Mouse anti β actin monoclonal antibody, by Proteintech (1 mentions)
Mouse anti flag monoclonal antibody, by Transgene (1 mentions)
Alkaline phosphatase, by Beyotime (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
7 protocols using horseradish peroxidase hrp conjugated goat anti mouse igg
1
PRRSV Protein Expression Analysis
2
Western Blot Validation of Schistosoma Proteins
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Two DEPs, SjGST and SjPDI, which were identified as upregulated in worms from water buffalo, were used to confirm the proteomic results by Western blotting. A pre-processed equivalent amount of protein was obtained from each sample, separated by 12% SDS-PAGE, and transferred to 0.45-μm pore size nitrocellulose membranes (Merck Millipore). The membranes were blocked overnight at 4°C with 5% non-fat milk in 0.05% Tween 20-PBS and incubated with monoclonal antibodies specific to mouse β-actin (Beyotime, China, 1:500 dilution), and mouse sera specific to SjPDI or SjGST (1:100), respectively. The membranes were washed three times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Beyotime, China, 1:2000 dilution). Moreover, the samples were detected using an automatic chemiluminescence imaging analysis system (Tanon, China).
3
Immunocytochemical Analysis of Recombinant Adenoviruses
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Immunocytochemistry was used to assess the protein expression of the recombinant adenoviruses. Briefly, the recombinant adenoviruses Ad2-p30, Ad2-p54, Ad2-p30-p54, Ad2-CD2v, and Ad2-p72-p72c were used to infect HEK-293 cell monolayers. The cells were fixed with 4% paraformaldehyde at 24 h after infection. The cells were blocked with 1× PBS containing 5% BSA at 37°C for 1 h and incubated with mouse anti-p30, anti-p54, anti-CD2v, and anti-p72 antibodies. These mouse antibodies against p30, p54, CD2v, or p72 were a gift from Prof Guangxia Gao. Following 3 washes, the cells were subsequently incubated for 1 h with a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Beyotime, China). Following washes as described above, the cells were stained with 3,3-diaminobenzidine tetrahydrochloride and then observed under a confocal microscope. Mock-infected cells were used as negative controls.
4
Enzyme-Linked Immunosorbent Assay for rp54 Protein
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Purified rp54 protein was diluted in carbonated coating buffer (pH = 9.6) at 2 µg/mL and used to coat flat-bottom polystyrene plates (100 µL/well) by overnight incubation at 4 °C. The plates were washed with PBST (0.05% Tween in PBS, v/v) three times before being blocked with 5% skimmed milk in PBS for 2 h at 37 °C. Then, the plates were washed another three times, and 100 µL hybridoma supernatants were loaded; positive sera from rp54 recombinant protein-immunized mice and negative sera from unimmunized mice (1:1000 dilution) were used as controls. After 60 min incubation at 37 °C and another washing step, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Beyotime Biotechnology Co., Ltd., Shanghai, China) (1:1000 dilution) was added for 45 min incubation at 37 °C. Following washing three times, a chromogenic substrate solution (TMB) (Beyotime Biotechnology Co., Ltd., Shanghai, China) was added and incubated for 10 min before being stopped with 2M H2SO4. The plates were read at 450 nm.
5
Protein Extraction and Western Blot
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The treated DEFs were lysed with Radio Immunoprecipitation Assay (RIPA ) lysis buffer (Thermo Fisher, USA) containing 1% phenylmethylsulfonyl fluoride (PMSF , Thermo Fisher, USA) for 30 min on ice. The cell lysates or proteins from the RNA pull-down assay were separated by 12% SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). Five percent skim milk was used to block the membranes for 2 h at room temperature. Mouse anti-flag monoclonal antibody (TransGen Biotech, Beijing, China) at a 1:5,000 dilution, mouse anti-E monoclonal antibody (prepared in our lab) at a 1:2,000 dilution, mouse anti-β-actin monoclonal antibody (Proteintech, Rosemont, IL) at a 1:2,000 dilution or mouse anti-GAPDH monoclonal antibody (Proteintech, Rosemont, IL) at a 1:2,000 dilution as the primary antibody was added to the membranes and cultured overnight at 4°C. Horseradish peroxidase (HRP )-conjugated goat anti-mouse IgG (Beyotime, Shanghai, China) at a 1:5,000 dilution was used as the secondary antibody. Finally, the Clarity Western ECL Substrate (Bio-Rad, CA, USA) was used for membrane imaging.
6
Propagation and Titration of Tembusu Virus in DF-1 Cells
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DF-1 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% inactivated fetal calf serum (FCS) in 5% CO2 at 37℃. TMUV JS804 was isolated by our laboratory and propagated in DF-1 cells as previously described [11 (link)]. All the experiments were approved by the ethical committee of Jiangsu Academy of Agricultural Sciences (approval No. SYXK-2015-0020). The titer of virus is calculated as TCID50 (50% tissue culture infective dose) according to the Reed-Muench method [26 ]. In brief, Serial 10-fold dilutions of the stock virus were inoculated into a monolayer of DF-1. After two-hour incubation at 37℃, inoculum was removed and cells were overlaid with a mixture of 1% agarose and cell culture media. Plaque formation was continuously observed daily for four days and TCID50 was calculated according to the method of Reed-Muench. TMUV-monoclonal antibody was generated and maintained by our laboratory as previously described [8 (link)]. Alkaline phosphatase and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG were purchased from the Beyotime Institute of Biotechnology (China). Anti-HSPA9 (ab171089, mouse) was purchased from Abcam (UK).
7
Enzyme-linked Immunosorbent Assay for rpI215L
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Each well of flat-bottom polystyrene plates was coated with 100 µL of purified rpI215L protein diluted carbonated coating buffer (pH 9.5) to a final concentration of 2 μg/mL at 4 °C overnight. The plates were washed three times with PBST (0.05% Tween in PBS, v/v) and blocked with 5% skimmed milk in PBS for 2 h at 37 °C. Hybridoma supernatants and ascites were diluted and added into the plates at 100 μg/well and incubated for 1 h at 37 °C. Positive serum obtained from rpI215L protein-immunized mice and negative serum from unimmunized mice (1:1000 dilution) were used as controls. After washing three times with PBST, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Beyotime Biotechnology Co., Ltd., Shanghai, China) (1:5000 dilution) was added as the secondary antibody and incubated for 45 min at 37 °C. Following another wash step, 50 µL of chromogenic substrate solution (TMB) (Beyotime Biotechnology Co., Ltd., Shanghai, China) was added and incubated for 10 min at 37 °C in the dark. 50 µL of 2 M H2SO4 was used to stop the reaction. OD values were measured at 450 nm using a microplate reader (BioTek Instruments, Winooski, VT, USA).
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