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A toolkit for expression of Strep-tagged enhanced green fluorescent protein concatemers in mammalian cells.

Authors
  • Zweifel, Aline1
  • Giehler, Susanne1
  • Nalaskowski, Marcus M2
  • 1 Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany. , (Germany)
  • 2 Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany. Electronic address: [email protected] , (Germany)
Type
Published Article
Journal
Analytical Biochemistry
Publisher
Elsevier
Publication Date
Dec 01, 2019
Volume
586
Pages
113430–113430
Identifiers
DOI: 10.1016/j.ab.2019.113430
PMID: 31521668
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Green fluorescent protein (GFP) and its variants are widely used tools in life sciences. Recently, we and others have used enhanced green fluorescent protein (EGFP) concatemers for determination of nuclear localization signal strength, as natural fluorescence standards and for mapping mobility in living cell nuclei. In this study, we present a molecular toolbox of Strep-tagged EGFP concatemers ranging from 1 to 12 subunits (Addgene plasmids #122488-122499). EGFP concatemers can be easily fused to targeting motifs of any origin by oligonucleotide ligation. Subsequently, we used liposomal transfection for transient expression of EGFP concatemers in eukaryotic cells. We have tested multiple protocols for further processing of the cells and recommend use of formalin or paraformaldehyde/methanol fixation. After usage of these protocols, we were able to detect concatemers by both GFP fluorescence microscopy and αStrep immunomicroscopy. In addition, we observed a more reliable detection of the StrepTag polypeptide (SA-WSHPQFEK) when using αStrepTag antibody instead of StrepTag binding protein. Summing up, we present a toolbox for expression of a wide range of Strep-tagged EGFP concatemers for multiple applications. By use of EGFP fluorescence and/or StrepTag polypeptide, the expressed concatemers can be easily detected in the cell. Copyright © 2019 Elsevier Inc. All rights reserved.

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