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

APEX-Gold: A genetically-encoded particulate marker for robust 3D electron microscopy

James Rae, Charles Ferguson, Nicholas Ariotti, Richard I. Webb, Han-Hao Cheng, James L. Mead, Jamie Riches, Dominic J.B. Hunter, Nick Martel, Joanne Baltos, Arthur Christopoulos, Nicole S. Bryce, Maria Lastra Cagigas, Sachini Fonseka, Edna C. Hardeman, Peter W. Gunning, Yann Gambin, View ORCID ProfileThomas Hall, Robert G. Parton
doi: https://doi.org/10.1101/2020.10.06.328831
James Rae
1The University of Queensland, Institute for Molecular Bioscience, Queensland 4072, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Charles Ferguson
1The University of Queensland, Institute for Molecular Bioscience, Queensland 4072, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nicholas Ariotti
2Mark Wainwright Analytical Centre, University of New South Wales, Sydney, NSW 2052, Australia
3School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Richard I. Webb
4The University of Queensland, Centre for Microscopy and Microanalysis, Queensland 4072, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Han-Hao Cheng
4The University of Queensland, Centre for Microscopy and Microanalysis, Queensland 4072, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
James L. Mead
4The University of Queensland, Centre for Microscopy and Microanalysis, Queensland 4072, Australia
8Division Microrobotics and Control Engineering, Department of Computing Science, University of Oldenburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jamie Riches
5Queensland University of Technology, Brisbane, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Dominic J.B. Hunter
1The University of Queensland, Institute for Molecular Bioscience, Queensland 4072, Australia
7EMBL Australia Node for Single Molecule Sciences, University of New South Wales, Sydney, NSW, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nick Martel
1The University of Queensland, Institute for Molecular Bioscience, Queensland 4072, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Joanne Baltos
6Monash Institute of Pharmaceutical Sciences, Monash University, Victoria, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Arthur Christopoulos
6Monash Institute of Pharmaceutical Sciences, Monash University, Victoria, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nicole S. Bryce
3School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Maria Lastra Cagigas
3School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sachini Fonseka
1The University of Queensland, Institute for Molecular Bioscience, Queensland 4072, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Edna C. Hardeman
3School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Peter W. Gunning
3School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Yann Gambin
7EMBL Australia Node for Single Molecule Sciences, University of New South Wales, Sydney, NSW, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Thomas Hall
1The University of Queensland, Institute for Molecular Bioscience, Queensland 4072, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Thomas Hall
Robert G. Parton
1The University of Queensland, Institute for Molecular Bioscience, Queensland 4072, Australia
4The University of Queensland, Centre for Microscopy and Microanalysis, Queensland 4072, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: R.Parton@imb.uq.edu.au
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Preview PDF
Loading

Abstract

Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy. Here we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal to noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins and cytoskeletal proteins. The method can be combined with different electron microscopic techniques including fast freezing and freeze substitution, focussed ion beam scanning electron microscopy, and electron tomography. The method allows detection of endogenously expressed proteins in genome-edited cells. We make use of a cell-free expression system to generate membrane particles with a defined quantum of an APEX-fusion protein. These particles can be added to cells to provide an internal standard for estimating absolute density of expressed APEX-fusion proteins.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
Back to top
PreviousNext
Posted October 06, 2020.
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.
APEX-Gold: A genetically-encoded particulate marker for robust 3D electron 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
APEX-Gold: A genetically-encoded particulate marker for robust 3D electron microscopy
James Rae, Charles Ferguson, Nicholas Ariotti, Richard I. Webb, Han-Hao Cheng, James L. Mead, Jamie Riches, Dominic J.B. Hunter, Nick Martel, Joanne Baltos, Arthur Christopoulos, Nicole S. Bryce, Maria Lastra Cagigas, Sachini Fonseka, Edna C. Hardeman, Peter W. Gunning, Yann Gambin, Thomas Hall, Robert G. Parton
bioRxiv 2020.10.06.328831; doi: https://doi.org/10.1101/2020.10.06.328831
Digg logo Reddit logo Twitter logo Facebook logo Google logo LinkedIn logo Mendeley logo
Citation Tools
APEX-Gold: A genetically-encoded particulate marker for robust 3D electron microscopy
James Rae, Charles Ferguson, Nicholas Ariotti, Richard I. Webb, Han-Hao Cheng, James L. Mead, Jamie Riches, Dominic J.B. Hunter, Nick Martel, Joanne Baltos, Arthur Christopoulos, Nicole S. Bryce, Maria Lastra Cagigas, Sachini Fonseka, Edna C. Hardeman, Peter W. Gunning, Yann Gambin, Thomas Hall, Robert G. Parton
bioRxiv 2020.10.06.328831; doi: https://doi.org/10.1101/2020.10.06.328831

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 (3506)
  • Biochemistry (7348)
  • Bioengineering (5324)
  • Bioinformatics (20266)
  • Biophysics (10020)
  • Cancer Biology (7744)
  • Cell Biology (11306)
  • Clinical Trials (138)
  • Developmental Biology (6437)
  • Ecology (9954)
  • Epidemiology (2065)
  • Evolutionary Biology (13325)
  • Genetics (9361)
  • Genomics (12587)
  • Immunology (7702)
  • Microbiology (19027)
  • Molecular Biology (7444)
  • Neuroscience (41049)
  • Paleontology (300)
  • Pathology (1230)
  • Pharmacology and Toxicology (2138)
  • Physiology (3161)
  • Plant Biology (6861)
  • Scientific Communication and Education (1273)
  • Synthetic Biology (1897)
  • Systems Biology (5313)
  • Zoology (1089)