Identification, Characterization, and In Vitro Culture of Highly Divergent Arenaviruses from Boa Constrictors and Annulated Tree Boas: Candidate Etiological Agents for Snake Inclusion Body Disease

Stenglein, Mark D.; Sanders, Chris; Kistler, Amy L.; Ruby, J. Graham; Franco, Jessica Y.; Reavill, Drury R.; Dunker, Freeland; DeRisi, Joseph L.

Western blotting. Tissues and cells were homogenized in ice-cold buffer containing 40 mM Tris (pH 7.4), 120 mM NaCl, 0.5% Triton X-100, 0.3% SDS, and Complete protease inhibitors (Roche). Samples were rotated for 30 min and then clarified by centrifugation for 10 min at 10,000 ϫ g at 4°C. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce). Five micrograms (cells) or 25 g (tissues) of total protein was fractionated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was probed with anti-NP polyclonal antiserum, which was detected with a fluorescently labeled secondary antibody. The blot was scanned on an Odyssey scanner (LiCor).