title: Metallothionein as a clonable tag for protein localization by electron microscopy of cells.
abstract: A benign, clonable tag for the localization of proteins by electron microscopy of cells would be valuable, especially if it provided labelling with high signal-to-noise ratio and good spatial resolution. Here we explore the use of metallothionein as such a localization marker. We have achieved good success with desmin labelled in vitro and with a component of the yeast spindle pole body labelled in cells. Heavy metals added after fixation and embedding or during the process of freeze-substitution fixation provide readily visible signals with no concern that the heavy atoms are affecting the behaviour of the protein in its physiological environment. However, our methods did not work with protein components of the nuclear pore complex, suggesting that this approach is not yet universally applicable. We provide a full description of our optimal labelling conditions and other conditions tried, hoping that our work will allow others to label their own proteins of interest and/or improve on the methods we have defined.
authors.affiliation: Department of Molecular, Cellular and Developmental, Biology University of Colorado, Boulder, Colorado, 80309-0347, U.S.A.
authors.forename: M K
authors.initials: MK
authors.lastname: Morphew
authors.affiliation: Department of Molecular, Cellular and Developmental, Biology University of Colorado, Boulder, Colorado, 80309-0347, U.S.A.
authors.forename: E T
authors.initials: ET
authors.lastname: O'Toole
authors.affiliation: Department of Molecular, Cellular and Developmental, Biology University of Colorado, Boulder, Colorado, 80309-0347, U.S.A.
authors.forename: C L
authors.initials: CL
authors.lastname: Page
authors.affiliation: Department of Molecular, Cellular and Developmental, Biology University of Colorado, Boulder, Colorado, 80309-0347, U.S.A.
authors.forename: M
authors.initials: M
authors.lastname: Pagratis
authors.affiliation: Department of Molecular, Cellular and Developmental, Biology University of Colorado, Boulder, Colorado, 80309-0347, U.S.A.
authors.forename: J
authors.initials: J
authors.lastname: Meehl
authors.affiliation: Department of Molecular, Cellular and Developmental, Biology University of Colorado, Boulder, Colorado, 80309-0347, U.S.A.
authors.forename: T
authors.initials: T
authors.lastname: Giddings
authors.affiliation: The Stowers Institute for Medical Research, Kansas City, Missouri, 64110 and Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, 66160, U.S.A.
authors.forename: J M
authors.initials: JM
authors.lastname: Gardner
authors.affiliation: Department of Chemistry, Colorado State University, Fort Collins, Colorado, U.S.A.
authors.forename: C
authors.initials: C
authors.lastname: Ackerson
authors.affiliation: The Stowers Institute for Medical Research, Kansas City, Missouri, 64110 and Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, 66160, U.S.A.
authors.forename: S L
authors.initials: SL
authors.lastname: Jaspersen
authors.affiliation: Department of Molecular, Cellular and Developmental, Biology University of Colorado, Boulder, Colorado, 80309-0347, U.S.A.
authors.forename: M
authors.initials: M
authors.lastname: Winey
authors.affiliation: Department of Molecular, Cellular and Developmental, Biology University of Colorado, Boulder, Colorado, 80309-0347, U.S.A.
authors.forename: A
authors.initials: A
authors.lastname: Hoenger
authors.affiliation: Department of Molecular, Cellular and Developmental, Biology University of Colorado, Boulder, Colorado, 80309-0347, U.S.A.
authors.forename: J R
authors.initials: JR
authors.lastname: McIntosh
mesh_terms: D003598:Cytoskeletal Proteins / Q000032:analysis* / Q000235:genetics; D003893:Desmin / Q000032:analysis*; D008668:Metallothionein / Q000737:chemistry / Q000378:metabolism; D008854:Microscopy, Electron / Q000379:methods; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D053758:Nanoparticles; D010750:Phosphoproteins / Q000032:analysis* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000235:genetics; D059629:Signal-To-Noise Ratio; D016610:Tissue Embedding; D016707:Tissue Fixation
chemical_list: D003598:Cytoskeletal Proteins; D003893:Desmin; D010750:Phosphoproteins; C432692:SPC42 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D008668:Metallothionein
keywords: Clonable labels; electron microscopy; immuno-electron microscopy; metallothionein; nanoparticles; protein labels