The F protein was analysed by sucrose gradient ultracentrifugation to examine the sedimentation properties of the F protein. RSV-infected cell monolayers were surface-biotinylated, and a detergent extract prepared using NP40 buffer. The clarified detergent extract was layered over a 5–30% sucrose gradient, which was then centrifuged at 210,000g at 4 oC. The gradient was separated into 12 individual fractions, and the presence of the F protein in these
Acetylsalicylic acid fractions determined by immunoblotting using MAb 169 (recognises the F1 subunit) ( Fig. 2A). The F1 subunit was located mainly in fractions 4 and 5, with little or no F protein being detected in the other fractions. These peak fractions were further examined by immunoprecipitation using MAb19, and the biotinylated protein profile determined ( Fig. 2B). This revealed the appearance of the 55 and 90 kDa protein species, which correspond in size to the F1 subunit and G protein. The immunoprecipitation assay from fraction 4 was analysed by immunoblotting with anti-G, which revealed the 90 kDa G protein ( Fig. 2C), thus confirming the identity of the co-migrating 90 kDa protein. As expected, no corresponding biotinylated protein bands were detected in fraction 3. These data demonstrated the co-migration of the F and G
proteins in the ultracentrifugation analysis, providing additional evidence for a protein complex involving these proteins.
