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 J. B. 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*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)
58 Downloads (Pure)

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 (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.
Original languageEnglish
Article numbere64630
Pages (from-to)1-17
Number of pages17
JournaleLife
Volume10
DOIs
Publication statusPublished - 2021
Externally publishedYes

Bibliographical note

Copyright the Author(s) 2021. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.

Fingerprint

Dive into the research topics of 'A robust method for particulate detection of a genetic tag for 3D electron microscopy'. Together they form a unique fingerprint.

Cite this