Figure 5

Conjugation of viral particles with Cy5 monoreactive dye. Purified TLP's of strain RRV were conjugated with different amounts of Cy5 monoreactive dye as described under Materials and methods. The reaction was stopped by Tris-HCl and labeled viruses were separated by gel filtration on G25 sepharose column. (A) Labeled and non-labeled viral particles were separated on 10% PAGE, and gel was visualized on Typhoon Trio to determine Cy5 - viral protein conjugation. (B) Same gel as shown in A was stained using silver nitrate. Viral proteins are identified by arrows. (C) MA104 cells grown in 96 well plates were infected with labeled and non labeled viral preparations, and 14 hours post infections cells were fixed and infected cells were detected using peroxydase immuno staining with anti-rotavirus polyclonal antibodies. Results are expressed as number of viral infectious focus forming units per ml. (D) Comparison of fluorophore labelled TLP's and DLP's (prepared by EDTA treatment), shown in red, with 100 nm Fluorosferes, shown in green, observed in confocal microscope.