HEK-293T cells were obtained from the ATCC. The Huh7.5 cells, the 9E10 antibody and J6/JFH1 recombinant virus (HCVcc) were generously provided by Dr. C. Rice (Rockefeller University). Dr. T. Wakita (National Institute of Infectious Diseases, Japan) and Dr. J. K. Ball (University of Nottingham) generously provided respectively the 2a JFH1 HCVcc and a panel of genotype 1–6 E1E2 constructs. The CD81 large extracellular loop fused to glutathione S-transferase was provided by Dr. S. Levy (Stanford University). Anti-CD81 (JS-81) was from BD Biosciences (Heidelberg, Germany) and anti-core C7-50 was from Abcam (Cambridge, UK).
Anti core c7 50
Manufactured by Abcam
Sourced in Germany
Anti-core C7-50 is a laboratory reagent used for research purposes. It is a monoclonal antibody that specifically binds to the core region of a target antigen. The core function of this product is to facilitate the detection and study of the target antigen in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Hek293t cell, by American Type Culture Collection (1 mentions)
Anti cd81 js 81, by BD (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti xrn1, by Fortis Life Sciences (1 mentions)
Pierce ecl western blot substrate, by Thermo Fisher Scientific (1 mentions)
3 protocols using anti core c7 50
1
HCV Entry and Replication Assay
2
Protein Extraction and Western Blotting
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Cells transfected for 3 days were washed with PBS once and lysed in RIPA buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and 1% Triton X-100) in the presence of cOmplete™, EDTA-free protease inhibitor cocktail (Roche) for 30 min on ice. Lysates were clarified to remove the non-soluble fraction by centrifugation at 14,000 rpm for 10 min at 4°C. Protein concentrations were measured by Bradford Assay and 50 μg of total protein lysate was mixed with 5X protein sample buffer containing reducing agent. Samples were separated on a 10% SDS-polyacrylamide gel, transferred to a PVDF membrane (Millipore), and blocked with 5% non-fat milk in PBS-T. The following primary antibodies were used to probe the membranes: anti-Core (C7-50) (Abcam, ab2740), anti-Actin (Sigma), and anti-XRN1 (Bethyl Lab A300-443A.) Immunoblots were developed using Pierce ECL Western Blot Substrate (Thermo Fisher) following the manufacturer’s suggested instructions.
3
Quantifying SARS-CoV-2 Infectivity in Huh7.5 Cells
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Infectivity was quantified as previously described (Mejer et al. 2020 ). Briefly, Huh7.5 cells were plated on 96-well plates at 6,000 cells/well and were infected 24 h post-seeding with serial dilutions of filtered culture supernatants, in triplicates. Cells were fixed 48 h post-infection with methanol, treated with 3 per cent hydrogen peroxide, and stained using anti-NS5A (9E10; a gift from C. M. Rice) (Lindenbach et al. 2005 (link)) and anti-Core (C7-50; Abcam) as primary antibodies. Secondary staining with anti-mouse antibody conjugated to horseradish peroxidase (GE Life Science), followed by incubation with 3,3ʹ-diaminobenzidine (DAB) substrate (Dako North America Inc), produced a color reaction in infected cells. Plates were scanned to count focus-forming units (FFU) using an Immunospot plate reader (CTL-Europe). Triplicate wells were averaged after removal of background, allowing calculation of the mean infectivity as Log10 FFU/ml.
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