Protocol: Optimised methodology for isolation of nuclei from leaves of species in the Solanaceae and Rosaceae families
1 Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry, Kaunas st 30, Babtai, LT-54333, Kaunas, Lithuania
2 Department of Agricultural Sciences, University of Helsinki, PO Box 27, FIN-00014 Helsinki, Finland
Plant Methods 2013, 9:31 doi:10.1186/1746-4811-9-31Published: 26 July 2013
In this study, a protocol is described for rapid preparation of an enriched, reasonably pure fraction of nuclear proteins from the leaves of tobacco (Nicotiana tabacum), potato (Solanum tuberosum) and apple (Malus domestica). The protocol gives reproducible results and can be carried out quickly in 2 hours. Tissue extracts clarified with filtration were treated with non-ionic detergent (Triton X-100) to lyse membranes of contaminating organelles. Nuclei were collected from a 60% Percoll layer of density gradient following low-speed centrifugation. Western blot analysis using antibodies to marker proteins of organelles indicated that the nuclear protein fractions were highly enriched and free or nearly free of proteins from the endoplasmic reticulum and chloroplasts.