title: Localizing Proteins in Fixed Giardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy.
abstract: Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes.
authors: Baptist College of Health Sciences, 1003 Monroe Avenue, Memphis, TN, 38104, USA.|Department of Biological Sciences, The University of Memphis, Memphis, TN, 38152, USA.
authors: Lilian
authors: L
authors: Nyindodo-Ogari
authors: Department of Biological Sciences, The University of Memphis, Life Sciences Bldg., Memphis, TN, 38152, USA.
authors: Steven D
authors: SD
authors: Schwartzbach
authors: Department of Biological Sciences, The University of Memphis, Life Sciences Bldg., Memphis, TN, 38152, USA.
authors: Omar
authors: O
authors: Skalli
authors: Department of Biological Sciences, The University of Memphis, Life Sciences Bldg. Room 409B, Memphis, TN, 38152, USA. cestrano@memphis.edu.
authors: Carlos E
authors: CE
authors: Estraño
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002734:Chlorophyll / Q000235:genetics* / Q000378:metabolism; D000077194:Chlorophyll A; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005055:Euglena / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D016829:Giardia lamblia / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D045332:Photosystem II Protein Complex / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D045332:Photosystem II Protein Complex; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D002734:Chlorophyll; D049452:Green Fluorescent Proteins; C037184:chlorophyll b; D000077194:Chlorophyll A
keywords: Fluorescence; Giardia; Immunofluorescence; Tissue culture; Transfection