A robust method for particulate detection of a genetic tag for 3D electron microscopy
James Rae,
Charles Ferguson,
Nicholas Ariotti,
Richard I Webb,
Han-Hao Cheng,
James L Mead,
James D Riches,
Dominic JB Hunter,
Nick Martel,
Joanne Baltos,
Arthur Christopoulos,
Nicole S Bryce,
Maria Lastra Cagigas,
Sachini Fonseka,
Marcel E Sayre,
Edna C Hardeman,
Peter W Gunning,
Yann Gambin,
Thomas E Hall,
Robert G Parton
Affiliations
James Rae
The University of Queensland, Institute for Molecular Bioscience, Queensland, Australia
Charles Ferguson
The University of Queensland, Institute for Molecular Bioscience, Queensland, Australia
Nicholas Ariotti
Mark Wainwright Analytical Centre, University of New South Wales, Sydney, Australia; School of Medical Sciences, University of New South Wales, Sydney, Australia
Richard I Webb
The University of Queensland, Centre for Microscopy and Microanalysis, Queensland, Australia
Han-Hao Cheng
The University of Queensland, Centre for Microscopy and Microanalysis, Queensland, Australia
James L Mead
The University of Queensland, Centre for Microscopy and Microanalysis, Queensland, Australia; Division Microrobotics and Control Engineering, Department of Computing Science, University of Oldenburg, Oldenburg, Germany
The University of Queensland, Institute for Molecular Bioscience, Queensland, Australia; EMBL Australia Node for Single Molecule Sciences, University of New South Wales, Sydney, Australia
Nick Martel
The University of Queensland, Institute for Molecular Bioscience, Queensland, Australia
Joanne Baltos
Monash Institute of Pharmaceutical Sciences, Monash University, Victoria, Australia
Arthur Christopoulos
Monash Institute of Pharmaceutical Sciences, Monash University, Victoria, Australia
The University of Queensland, Institute for Molecular Bioscience, Queensland, Australia; The University of Queensland, Centre for Microscopy and Microanalysis, Queensland, Australia
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 (EM). 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 EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.
