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Hrp labelled secondary antibody

Manufactured by Jackson ImmunoResearch
Sourced in United States

HRP-labelled secondary antibodies are used to detect and visualize target proteins in various immunodetection techniques, such as Western blotting, ELISA, and immunohistochemistry. These antibodies are conjugated with the enzyme horseradish peroxidase (HRP), which catalyzes a colorimetric or chemiluminescent reaction, allowing the visualization of the target protein.

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6 protocols using hrp labelled secondary antibody

1

Western Blot Analysis of Protein Levels

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Total protein was extracted from both the tissues and cells following the instructions of the RIPA lysis buffer (Sigma, United States). Next, total protein concentration was measured using the BCA assay (Beyotime, China). Total proteins (30 ug) were separated on a 4–12% polyacrylamide gel by SDS-PAGE and electrophoretically transferred onto PVDF membranes, subsequently sealed with 5% BSA. The membranes were incubated with diluted primary antibodies to EZH2 (1:2,000 dilution, Abcam), RND3 (1:1,000 dilution, Abcam), IQCG (1:1,000 dilution, Abcam) overnight at 4 °C. The membrane was then further incubated with HRP-labelled secondary antibodies (Jackson, United States) at room temperature for 1 h. Finally, protein bands were analyzed and quantified using the ImageJ software.
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2

Protein Extraction and Western Blot Analysis

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Cells were lysed in a buffer containing 50 mM HEPES pH 7.3, 0.15 mM EDTA pH 7.9, 4 mM EGTA, 150 mM NaCl and 1% Triton X-100 supplemented with a cocktail of protease inhibitors (aprotinin, leupeptin, pepstatin-A, and pefabloc, 10 μg/ml) for 30 min at 4°C. After centrifugation for 15 min at 8000 rpm (4°C), protein concentration was determined using a BCA kit (Thermo Scientific). Protein levels were normalized to 100 μg per sample and resuspended with 4× laemmli sample buffer (Bio-Rad) before boiling (5 min). Proteins were separated by SDS-PAGE using precast 4–20% gels (Bio-Rad) and then transferred onto a nitrocellulose membrane. Membranes were then blocked with Tris-Tween buffered solution (TTBS; 10 mM Tris, 200 mM NaCl, 0.05% Tween 20, pH 7.4) containing 5% non-fat dry milk for 1 h at RT and then incubated with primary antibodies (rabbit anti-Rnd2, 1/250, Proteintech, 13844–1-AP; mouse anti-beta actin, 1/2000, Sigma, A5316) overnight at 4°C. HRP-labelled secondary antibodies (Jackson ImmunoResearch) were incubated the following day for 1 h at RT.
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3

Quantitative Western Blot Analysis of Muscle Proteins

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Western blot analysis was performed on muscle homogenates as previously described [16 (link)]. Briefly, after electrophoretic separation on a 4–20% gradient acrylamide gel (Stain-free precast gel, Biorad, France) and electrotransfer to Immobilon P (Biorad, France), the membrane was incubated with primary antibodies and then HRP-labelled secondary antibodies (Jackson ImmunoResearch Laboratories). Signal quantification was performed using a ChemiDoc Touch apparatus (Biorad, France) and the Image Lab software (Biorad). The amount of the chosen protein in each sample was corrected for differences in loading using either the amount of myosin, GAPDH or the total amount of proteins using the stain free system from Biorad, and normalized to the amount of the same protein present in the control, set to 100% as described previously [15 (link)]. Ten human controls (muscle biopsy from individuals non-affected by neuromuscular disease) of different age have been used, from 3.5 to 64 years. For each sample (mouse and human), 2–3 Western blots have been performed, and the value for each sample corresponds to the mean ± SEM of the different Western blots.
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4

Antibody and Reagent Catalog for Virus Research

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The following antibodies, beads, and dyes were obtained commercially: HCV core (clone 7–50, sc-57800), HA (clone Y-11, sc-805-G), PABPC1 (clone 10E10, sc-32318), MOV10 (clone B-3, sc-515722), calnexin (c-20, sc-6465), TIA1 (c-20, sc-1751) (all Santa Cruz Biotechnology), G3BP1 (Clone 23/G3BP, 611127, BD Biosciences), PLIN2 (GP40, Progen; ab52356, abcam), HCV NS3 (ab65407, abcam), HCV NS5A (HCM-131-5, IBT), FLAG (F7425), FLAG (F1804), HA (H6908), tubulin (clone B5-1-2, T6074), anti-FLAG M2 affinity gel (A2220), anti-HA affinity gel (HA-7, A2095), recombinant protein A (15918–014) and protein G (15920–010) agarose beads (all Sigma), L1ORF1p (clone 4H1, MABC1152, Merck), YB-1 (ab12148, abcam), HRP-labelled secondary antibodies (Jackson ImmunoResearch), HRP-labelled TrueBlot secondary antibodies (Rockland Immunochemicals), Alexa488-, Alexa555-, and Alexa647-conjugated secondary antibodies (all donkey, IgG (H+L)), BODIPY493/503 (D-3922), BODIPY 655/676 (B-3932) (all Life Technologies), Hoechst33342 (Thermo Fisher). L1ORF1p antibody #984 was described previously [33 (link)]. Oligonucleotides and PCR primer were purchased from Sigma. Restriction enzymes for molecular cloning were obtained from NEB, other enzymes from Thermo Fisher. Unless stated otherwise, chemicals were purchased from Sigma or Applichem and cell culture reagents from Gibco/Thermo Fisher.
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5

Western Blot Analysis of Protein Expression

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Samples were homogenized in M-PER® Mammalian Protein Extraction Reagent lysis buffer (Thermo, MI, USA) containing protease inhibitor cocktail and 0.5 M EDTA (Thermo, MI, USA) (1:100 dilution). The supernatant was collected after centrifugation at 12000 rpm for 30 min at 4 °C. The protein concentration was determined using the BCA Protein Assay Kit (Thermo, MI, USA). The protein samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The membranes were blocked with 5% nonfat milk at room temperature for 1 h and then incubated with anti-TTF1 (1:500, Abcam, MA, USA), anti-Kiss1 (1:400, Abcam, MA, USA) or anti-β-tubulin antibody (1:1000, CST, MA, USA) at 4 °C overnight. Subsequently, the membranes were rinsed three times with Tris-buffered saline with 0.1% Tween 20 (TBST) every 10 min and probed with an HRP-labelled secondary antibody (1:10000, Jackson, PA, USA) at room temperature for 1 h. After three additional rinses with TBST, the membranes were visualized using the ECL system. The grey values of the protein bands were analysed using ImageJ software.
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6

Genomic DNA Methylation Analysis

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Dot blot assay was used to analyze genomic DNA that was isolated from P. torridus cultures as described above. For this, 1 μg genomic DNA was digested with EcoRI and then denatured with 0.3 N sodium hydroxide for 30 min. This was followed by neutralization with 1 M ammonium acetate before spotting on Hybond N+ nylon membrane (GE Healthcare, United States) using the Bio-Dot apparatus (Bio-Rad Laboratories, United States). The membrane was baked at 80°C for 2 h before probing with anti-m6A antibody (Sigma Aldrich, Cat. No. ABE572) or anti-m5C antibody (EpiGentek, Cat.No. A-1014-010). This was done by blocking the baked dot blots with 10% skim milk (Difco Laboratories) for an hour at room temperature, washing the blots twice with 1X PBS-T, and then incubating them overnight at 4°C with the anti-m6A or anti-m5C antibodies (1:1000 dil in 1X PBS). Following three washes with 1X PBS-T the blots were incubated with HRP-labelled secondary antibody (1:10000 dil, Jackson Laboratory, United States) for an hour and developed using a chemiluminescence method.
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