A robust method for particulate detection of a genetic tag for 3D electron microscopy

eLife

Yann Gambin¹  Nicholas Ariotti²  Joanne Baltos³  Arthur Christopoulos³  James D Riches⁴  Edna C Hardeman⁵  Peter W Gunning⁵  Nicole S Bryce⁵  Maria Lastra Cagigas⁵  Richard I Webb⁶  Han-Hao Cheng⁶  Marcel E Sayre⁶  James L Mead⁷  James Rae⁸  Sachini Fonseka⁸  Thomas E Hall⁸  Charles Ferguson⁸  Nick Martel⁸  Dominic JB Hunter⁹  Robert G Parton^1,10 

[1] EMBL Australia Node for Single Molecule Sciences, University of New South Wales, Sydney, Australia;Mark Wainwright Analytical Centre, University of New South Wales, Sydney, Australia;School of Medical Sciences, University of New South Wales, Sydney, Australia;Monash Institute of Pharmaceutical Sciences, Monash University, Victoria, Australia;Queensland University of Technology, Queensland, Australia;School of Medical Sciences, University of New South Wales, Sydney, Australia;The University of Queensland, Centre for Microscopy and Microanalysis, Queensland, Australia;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;The University of Queensland, Institute for Molecular Bioscience, Queensland, Australia;EMBL Australia Node for Single Molecule Sciences, University of New South Wales, Sydney, Australia;The University of Queensland, Institute for Molecular Bioscience, Queensland, Australia;The University of Queensland, Centre for Microscopy and Microanalysis, Queensland, Australia;

关键词: APEX;    electron microscopy;    particulate marker;    3D-electron microscopy;    genetically encoded;    Mouse;   

DOI  :  10.7554/eLife.64630

来源: eLife Sciences Publications, Ltd

【 摘 要 】

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.

【 授权许可】

CC BY