Identification of NL63-ORF 3 protein as a structural viral protein by sucrose gradient ultracentrifugation. Viral supernatant was purified via subsequently centrifugation on two discontinuous and one continuous sucrose gradients of 20% to 60% (w/v) sucrose. The continuous cushion was divided into ten fractions as indicated in part (A). After centrifugation of each fraction through 20% sucrose cushions, the resulting pellets were analyzed for infectious particles by plaque assays. Resulting virus titers are indicated on the 20 Y-axis in part (A). (B), fractions 4-8 were subjected to Western blot analysis using specific rabbit antibodies against ORF 3, M and N protein (1:3000; 1:250,000 and 1:24,000, respectively). To exclude cellular contaminations in the fractions a Western blot using mouse-anti-actin (1:2,000) was performed. Note the colocalization of the ORF 3 protein in the same gradients as the known structural proteins M and N.