Native Electrophoresis and Western Blot Analysis: Method and Applications
Native electrophoresis and western blot analysis (NEWeB) has been developed for the study of plant virus characteristics, among others, virus particle–protein interactions, electrophorotype formation, and strain separation. The method is based on the property of electrophoretic mobility of virus particles (VP) and proteins and combines the analytical capacity of electrophoresis with the specificity of western blot. One of its advantages is that it deals with entire VP that can be studied in cause and effect or in time-interval experiments. Some of the most interesting approaches include VP structural studies, VP interaction with host or viral proteins, and also the characterization of VP–protein complexes. In this protocol, NEWeB is used to demonstrate the interaction of Plum pox virus particles with the helper component, a virus encoded protein. It is expected that the method could be used in analogous studies of other viruses or large protein complexes, where similar principles apply.
Key wordsNative electrophoresis NEWeB Virus particle–protein interaction Helper component HCPro Coat protein Plum pox virus Potyvirus
We thank Prof. E. Maiss for providing PPV CP and HC specific antibodies and M. Bastaki for help during her rotation. This work was partly supported by the Greek Ministry of Development (Project EPET II, 453) and by the INTAS 989 program of the EC.
- 1.Levy A.J., Fraenkel-Conrat H., & Owens A.R. (1994) Virology (Prentice Hall International Limited, London UK).Google Scholar
- 2.Gibbs A., & Harrison B.D. (1976) Plant Virology: The Principles (London, Edward Arnold).Google Scholar
- 3.Tsagris M., Liu., B., Tzortzakaki, S., & Tabler, M. (1993) Construction of a full length cDNA clone of an aphid transmissible PPV strain from Greece p66–21. IX International Congress of Virology.Google Scholar
- 6.Manoussopoulos, I. N., Maiss, E., & Tsagris, E. (1996). Detection of virus particle-helper component complexes in Nicotiana benthamiana plants infected with plum pox virus. PW37-8. Jerusalem, Israel, Xth International Congress of virology.Google Scholar
- 8.Manoussopoulos, I. N., Maiss, E., & Tsagris, M. (2000) Native electrophoresis and Western blot analysis (NEWeB): a method for characterization of different forms of potyvirus particles and similar nucleoprotein complexes in extracts of infected plant tissues. J Gen Vir 81:2295–2298.Google Scholar