Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells.
Transient expression of fluorescent fusion proteins in plant cells has dramatically facilitated our study of newly identified genes and proteins. This protocol details an in vivo transient expression system to study the subcellular localization and dynamic associations of plant proteins using protoplasts freshly prepared from Arabidopsis or tobacco BY-2 suspension cultured cells. The method relies on the transformation of DNA constructs into protoplasts via electroporation. The whole protocol is comprised of three major stages: protoplast generation and purification, transformation of DNA into protoplasts via electroporation and incubation of protoplasts for protein analysis. Similar to stably transformed cell lines, transformed protoplasts are compatible with protein localization studies, pharmaceutical drug treatment and western blot analysis. This protocol can be completed within 11-24 h from protoplast production to protein detection.
Miao
Yansong
Y
Department of Biology and Molecular Biotechnology Program, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.
Jiang
Liwen
L
D017360:Arabidopsis / Q000235:genetics; D002478:Cells, Cultured; D018274:Electroporation / Q000379:methods; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D008164:Luminescent Proteins / Q000032:analysis*; D008856:Microscopy, Fluorescence; D010940:Plant Proteins / Q000032:analysis*; D030821:Plants, Genetically Modified / Q000378:metabolism; D011523:Protoplasts / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis*; D014026:Nicotiana / Q000235:genetics; D014170:Transformation, Genetic; D014617:Vacuoles / Q000378:metabolism
D008164:Luminescent Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins