CUED Publications database

Quantification of very low-abundant proteins in bacteria using the HaloTag and epi-fluorescence microscopy

Lepore, A and Taylor, H and Landgraf, D and Okumus, B and Jaramillo-Riveri, S and McLaren, L and Bakshi, S and Paulsson, J and Karoui, ME (2019) Quantification of very low-abundant proteins in bacteria using the HaloTag and epi-fluorescence microscopy. Scientific Reports, 9.

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Abstract

© 2019, The Author(s). Cell biology is increasingly dependent on quantitative methods resulting in the need for microscopic labelling technologies that are highly sensitive and specific. Whilst the use of fluorescent proteins has led to major advances, they also suffer from their relatively low brightness and photo-stability, making the detection of very low abundance proteins using fluorescent protein-based methods challenging. Here, we characterize the use of the self-labelling protein tag called HaloTag, in conjunction with an organic fluorescent dye, to label and accurately count endogenous proteins present in very low numbers (<7) in individual Escherichia coli cells. This procedure can be used to detect single molecules in fixed cells with conventional epifluorescence illumination and a standard microscope. We show that the detection efficiency of proteins labelled with the HaloTag is ≥80%, which is on par or better than previous techniques. Therefore, this method offers a simple and attractive alternative to current procedures to detect low abundance molecules.

Item Type: Article
Subjects: UNSPECIFIED
Divisions: Div F > Control
Depositing User: Cron Job
Date Deposited: 05 Aug 2019 20:01
Last Modified: 10 Dec 2019 02:10
DOI: 10.1038/s41598-019-44278-0