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Horseradish peroxidase hrp conjugated anti rabbit igg

Manufactured by Beyotime
Sourced in United States

Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG is a secondary antibody used in various immunoassay techniques. It consists of an anti-rabbit IgG antibody covalently linked to the enzyme horseradish peroxidase. The HRP label allows for the detection and visualization of target proteins recognized by the primary rabbit antibody.

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4 protocols using horseradish peroxidase hrp conjugated anti rabbit igg

1

Deglycosylation of Influenza HA Protein

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Deglycosylation was performed using PNGase F or Endo H enzyme (New England BioLabs, MA) as previously described (57 (link)). In brief, virus samples were predenatured and then treated with or without the PNGase F for 15 min at 50°C or Endo H enzyme for 1 h at 37°C according to the manufacturer's instructions. Then samples were heated at 100°C for 10 min and then separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), and subsequently incubated with a primary antibody specific for H5N1 influenza HA (1:2,000, 11689-T54, Sino Biological). The secondary antibody used was horseradish peroxidase (HRP)-conjugated antirabbit IgG (1:10,000, A0208, Beyotime). HRP was detected using a Western Lightning chemiluminescence kit (Amersham Pharmacia, Freiburg, Germany) according to the manufacturer's protocols.
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2

Replication-Competent HBV Genome Construct

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The replication-competent HBV genome construct pREP-HBV1.2 (pHBV-1.2) was provided by Lin Xu. Plasmids were transfected into HepG2 or HL-7702 cells using Lipofectamine 3000 (Invitrogen, Thermo Fisher, USA) according to the manufacturer’s instructions. Anti-IFIT3 antibody (catalog no. ab236243) was purchased from Abcam (USA); anti-STAT2/phospho-STAT2, anti-Mx1, anti-PKR, anti-OAS1, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were obtained from Cell Signaling Technologies (USA); and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG antibodies were purchased from Beyotime Biotechnology (China).
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3

Ovarian Cancer Cell Line Characterization

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The mTOR inhibitor, rapamycin, was purchased from Calbiochem (San Diego, CA, USA). Propidium iodide (PI) was from Sigma. Anti-AE2 from Affinity (Cincinnati, OH, USA).The antibodies against p16, CDK4 and Cyclin D1 were from Santa Cruz Biotechnology (Santa Cruz, CA), and the antibodies against p-AKT (Ser473), AKT, p-mTOR (Ser2448), mTOR, p70S6K1, phospho-p70S6K1 (Thr389) and GAPDH were from Cell Signaling Technology (Beverly, MA). The horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-mouse IgG were from Beyotime. The human ovarian cancer cell lines OVCAR3, SK-OV-3, HO-8910, COC1 and A2780 (American Type Culture Collection; Rockville, MD, USA) were maintained in RPMI 1640 (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (FBS, Hyclone) and penicillin/streptomycin, and cultured at 37 °C in a 5% CO2 incubator.
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4

Protein Expression Analysis in Cells

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In short, cells were washed with cold PBS three times and lysed with radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors for 30 min on ice. The total protein concentration was quantified by a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Samples were separated by SDS/polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes (Millipore). Then the membranes were blocked with 5% fat-free milk dissolved in TBST (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, and 0.05% Tween 20) at room temperature for 1 h and incubated with primary antibodies against GAPDH (1:1,000, CW0100M; CWBIO), NAT10 (1:1,000, 13365-1-AP; Proteintech), RUNX2 (1:1,000 dilution, sc-390351; Santa Cruz), and Osterix (1:1,000, ab209484; Abcam) overnight at 4°C. After that, the membranes were incubated with the appropriate secondary antibody, either horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:3,000; Beyotime) or anti-mouse IgG (1:3,000; Beyotime), at room temperature for 1 h and visualized with chemiluminescent HRP substrate (Millipore, Billerica, MA, USA). ImageJ software was used to analyze the band intensities.
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