Fluorescent labeling of COS-7 expressing SNAP-tag fusion proteins for live cell imaging

Journal of Visualized Experiments : JoVE
Christopher R Provost, Luo Sun

Abstract

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O(6)-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

References

May 8, 2013·Biologicals : Journal of the International Association of Biological Standardization·Nur Hidayah Hairul BaharaTheam Soon Lim
Aug 2, 2012·BMC Plant Biology·Madlen SchillerIris Steinebrunner
Jun 17, 2015·Bioconjugate Chemistry·S Hoehnel, M P Lutolf

Citations

Jan 17, 2009·Nature Chemical Biology·Kai Johnsson

Related Concepts

Guanine
Cricetulus
Covalent Interaction
Transfection
Derivatives
Chimeric Proteins, Recombinant
COS-7 Cells
Chinese Hamster Ovary Cell
Immunofluorescence Microscopy
DNA Repair Methyltransferase II

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