Detection of Endogenous Nuclear Proteins in Plant Cells: Localizing Nuclear Matrix Constituent Proteins (NMCPs), the Plant Analogs of Lamins.
At present, two complementary approaches are used for in situ protein visualization in plant nuclei. Imaging of transformed fluorescent proteins is the election tool for the analysis of protein movement and interaction. However, this methodology presents several drawbacks for the identification/localization of endogenous nuclear factors, such as over-expression or mislocalization of transformed proteins. In contrast, immunocytochemistry with specific antibodies represents a powerful tool for the localization of endogenous nuclear proteins at their "native" nuclear sub-compartments. In plant cells, the cell wall hampers antibody accessibility during immunocytochemical analysis thereby reducing the effectivity of the technique, particularly in the case of lowly expressed proteins. To overcome this problem in nuclear protein immunodetection, we developed a method based on the in vitro incubation of isolated nuclei with specific antibodies followed by imaging by confocal fluorescence or electron microscopy. Here we describe the application of this methodology to the localization of Nuclear Matrix Constituent Proteins (NMCP), the plant analogs of lamins, of the monocot Allium cepa, using antibodies raised against highly conserved regions of the proteins.
Department of Cell and Molecular Biology, Biological Research Center, CSIC, Ramiro de Maeztu 9, 28040, Madrid, Spain.
Malgorzata
M
Ciska
Department of Cell and Molecular Biology, Biological Research Center, CSIC, Ramiro de Maeztu 9, 28040, Madrid, Spain. smoreno@cib.csic.es.
Susana Moreno Díaz
SM
de la Espina
D007150:Immunohistochemistry / Q000379:methods; D034882:Lamins / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D016253:Microscopy, Immunoelectron / Q000379:methods; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*
D034882:Lamins; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; D010940:Plant Proteins
Allium cepa; Immunoelectron microscopy; Immunofluorescence confocal microscopy; NMCP proteins; Nuclear isolation; Plant cells; Pre-embedding labeling