Visualizing dsRNA and p14 Proteins

Diluted dsRNA (E200 synthesized as described previously [21 (link)]) or purified p14 were applied to a dry Hybond-N+ positively charged nylon membrane (Amersham) and crosslinked with 100 µJ/cm² UV light using a Stratalinker 1800 (Stratagene). The membrane was blocked in 5% skim milk in TBS for 1 h, incubated with J2 antibody (SCICONS) diluted 1:2000 in 1% BSA in TBS for 2 h, followed by HRP-conjugated anti-mouse secondary antibody (Sigma, St. Louis, MO, USA) diluted 1:5000 in 1% skim milk in TBS for 1 h. An ECL solution of luminol, 4-IPBA and H₂O₂ in 100 mM Tris, as described [22 (link)], was added prior to X-ray film exposures.

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Hare D.N., Murdza T., Collins S., Schulz K., Mukherjee S., de Antueno R., Janssen L., Duncan R, & Mossman K.L. (2023). Differential Cellular Sensing of Fusion from within and Fusion from without during Virus Infection. Viruses, 15(2), 301.

Publication 2023

Hybond-N+ positively charged nylon membrane J2 antibody HRP-conjugated anti-mouse secondary antibody

4 ipba Anti antibody Antibody Dsrna H2o2 Luminol Membrane Milk Mouse Nylon Tris Uv light X ray film

Corresponding Organization : McMaster University

Other organizations : St. Joseph’s Healthcare Hamilton, Dalhousie University

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Variable analysis

independent variables

Diluted dsRNA (E200 synthesized as described previously [21 (link)])
Purified p14

dependent variables

Signal intensity measured by X-ray film exposures

control variables

Dry Hybond-N+ positively charged nylon membrane (Amersham)
UV light exposure at 100 µJ/cm^2 using a Stratalinker 1800 (Stratagene)
Blocking in 5% skim milk in TBS for 1 h
Incubation with J2 antibody (SCICONS) diluted 1:2000 in 1% BSA in TBS for 2 h
Incubation with HRP-conjugated anti-mouse secondary antibody (Sigma, St. Louis, MO, USA) diluted 1:5000 in 1% skim milk in TBS for 1 h
Addition of ECL solution of luminol, 4-IPBA and H2O2 in 100 mM Tris prior to X-ray film exposures

positive control

Not explicitly mentioned

negative control

Not explicitly mentioned

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