Bats Possess Unique Variants of the Antiviral Restriction Factor Tetherin

Joshua A. Hayward; Mary Tachedjian; Adam Johnson; Tamsin B. Gordon; Jie Cui; Glenn A. Marsh; Michelle L. Baker; Lin-Fa Wang; Gilda Tachedjian

x g for 10 min to separate the samples into two fractions; the upper, detergent-depleted fraction containing hydrophilic, soluble proteins and the lower, detergent-enriched fraction containing hydrophobic, transmembrane-and GPI-anchored proteins, including tetherin. The upper fractions of each sample were carefully aspirated by pipetting and discarded. The protein content of each sample was then purified and concentrated by trichloracetic acid (TCA) precipitation. A 25% volume of 100% (w/v) TCA was added to each sample (i.e. for sample volumes estimated to be 200 μl, 50 μl of TCA was added) and incubated on ice for 10 min. Samples were centrifuged at 20,000 x g for 5 min. The supernatant of each sample was then discarded and the protein pellets were washed with 200 μl of ice cold acetone followed by centrifugation at 20,000 x g for 5 min. The acetone wash was repeated once. The protein pellet was then dried on at 95°C for 5 min. Protein pellets were then resuspended in 20 μl of 4X SDS-PAGE sample buffer (Sodium dodecyl sulphate polyacrylamide gel electrophoresis sample buffer; 40% glycerol, 8% SDS, 240 mM Tris-Cl pH 6.8, 5% β-mercaptoethanol, 0.04% bromophenol blue).