Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

An icosahedral virus as a fluorescent calibration standard: a method for counting protein molecules in cells by fluorescence microscopy

John M. Murray
doi: https://doi.org/10.1101/088617
John M. Murray
Department of Biology, Indiana University, Bloomington, IN, 47405, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Preview PDF
Loading

Abstract

The ability to replace genes coding for cellular proteins with DNA that codes for fluorescent protein-tagged versions opens the way to counting the number of molecules of each protein component of macromolecular assemblies in vivo by measuring fluorescence microscopically. Converting fluorescence to absolute numbers of molecules requires a fluorescent standard whose molecular composition is known precisely. In this report the construction, properties, and mode of using a set of fluorescence calibration standards are described. The standards are based on an icosahedral virus engineered to contain exactly 240 copies of one of seven different fluorescent proteins. Two applications of the fluorescent standards to counting molecules in the human parasite Toxoplasma gondii are described. Methods for improving the preciseness of the measurements and minimizing potential inaccuracies are emphasized.

Lay Abstract A broad goal of modern biology is to understand how the machines within living cells work. It is nowadays routine to identify the individual protein components of a machine, but not yet straightforward to tell how many copies of each component are needed to build a functional assembly. In many types of cells it is now possible to substitute for the native proteins within cells altered versions that are fluorescent. If one knew how much fluorescence is generated by a single molecule of the altered protein, then one could use a light microscope to count the number of copies of the protein in a cellular machine by simply measuring the total fluorescence coming from that part of the cell. This paper describes the construction and methods for using a set of fluorescent virus particles that can be used to determine how much fluorescence is contributed by one molecule of fluorescent protein. The virus particles were chosen for this role because the particular icosahedral symmetry of their structure guarantees that each particle contains exactly 240 copies of one fluorescent protein.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Back to top
PreviousNext
Posted February 18, 2017.
Download PDF
Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
An icosahedral virus as a fluorescent calibration standard: a method for counting protein molecules in cells by fluorescence microscopy
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
An icosahedral virus as a fluorescent calibration standard: a method for counting protein molecules in cells by fluorescence microscopy
John M. Murray
bioRxiv 088617; doi: https://doi.org/10.1101/088617
Digg logo Reddit logo Twitter logo Facebook logo Google logo LinkedIn logo Mendeley logo
Citation Tools
An icosahedral virus as a fluorescent calibration standard: a method for counting protein molecules in cells by fluorescence microscopy
John M. Murray
bioRxiv 088617; doi: https://doi.org/10.1101/088617

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Cell Biology
Subject Areas
All Articles
  • Animal Behavior and Cognition (4118)
  • Biochemistry (8825)
  • Bioengineering (6529)
  • Bioinformatics (23481)
  • Biophysics (11802)
  • Cancer Biology (9221)
  • Cell Biology (13335)
  • Clinical Trials (138)
  • Developmental Biology (7442)
  • Ecology (11422)
  • Epidemiology (2066)
  • Evolutionary Biology (15171)
  • Genetics (10449)
  • Genomics (14054)
  • Immunology (9184)
  • Microbiology (22186)
  • Molecular Biology (8821)
  • Neuroscience (47615)
  • Paleontology (350)
  • Pathology (1431)
  • Pharmacology and Toxicology (2492)
  • Physiology (3736)
  • Plant Biology (8087)
  • Scientific Communication and Education (1438)
  • Synthetic Biology (2222)
  • Systems Biology (6042)
  • Zoology (1254)