Lookup NU author(s): Dr Yulia Yuzenkova,
Professor Nikolay Zenkin
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
Studying the structure and dynamics of proteins in live cells is essential to understanding their physiological activities and mechanisms, and to validating in vitro characterization. Improvements in labeling and imaging technologies are starting to allow such in vivo studies; however, a number of technical challenges remain. Recently, we developed an electroporation-based protocol for internalization, which allows biomolecules labeled with organic fluorophores to be introduced at high efficiency into live E. coli (Crawford et al. in Biophys J 105 (11): 2439-2450, 2013). Here, we address important challenges related to internalization of proteins, and optimize our method in terms of (1) electroporation buffer conditions; (2) removal of dye contaminants from stock protein samples; and (3) removal of non-internalized molecules from cell suspension after electroporation. We illustrate the usability of the optimized protocol by demonstrating high-efficiency internalization of a 10-kDa protein, the. subunit of RNA polymerase. Provided that suggested control experiments are carried out, any fluorescently labeled protein of up to 60 kDa could be internalized using our method. Further, we probe the effect of electroporation voltage on internalization efficiency and cell viability and demonstrate that, whilst internalization increases with increased voltage, cell viability is compromised. However, due to the low number of damaged cells in our samples, the major fraction of loaded cells always corresponds to non-damaged cells. By taking care to include only viable cells into analysis, our method allows physiologically relevant studies to be performed, including in vivo measurements of protein diffusion, localization and intramolecular dynamics via single-molecule Forster resonance energy transfer.
Author(s): Sustarsic M, Plochowietz A, Aigrain L, Yuzenkova Y, Zenkin N, Kapanidis A
Publication type: Article
Publication status: Published
Journal: Histochemistry and Cell Biology
Print publication date: 01/07/2014
Online publication date: 03/04/2014
Acceptance date: 13/03/2014
Date deposited: 01/10/2014
ISSN (print): 0948-6143
ISSN (electronic): 1432-119X
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