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Protein Labeling And Localization In Cells Using A Self Labeling Protein Tag
Author(s) -
Gedge L.,
Muentener K.,
Gibbs T.,
Brecht A.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a843-c
Subject(s) - green fluorescent protein , fusion protein , microbiology and biotechnology , chemistry , internalization , protein tag , biochemistry , target protein , covalent bond , biophysics , cell , biology , recombinant dna , gene , organic chemistry
Autofluorescent proteins like GFP have become a basic tool for todays cell biologist. We will discuss the utility of an alternative approach, based on covalently self‐labeling protein tags. One of these tags is the SNAP‐tag which is based on the human protein AGT. This tag reacts covalently with a wide range of substrates that are based on benzylguanine (BG). Three cell permeable fluorescent substrates are available so far which allow covalent labeling of SNAP‐tag fusion proteins inside living cells with blue, green, and red fluorophores. In addition non‐cell permeable fluorescent labels exist. The applicability of the concept for cellular studies will be discussed using the following examples: 1) Labeling of SNAP‐PKC‐gamma fusion protein in CHO‐cells and proof of intracellular redistribution upon stimulation with a phorbol ester. 2) Exclusive extracellular labeling of SNAP‐NK1 expressed in CHO‐cells and its internalization upon stimulation with substance P. 3) The sequential (pulse chase) labeling of a SNAP‐actin with two different colors inside living cells with the clear identification of resting and of extending actin filaments. A further result of interest is the fixation stability of labels introduced using self labeling protein tags, and the possibility of labeling a tag in fixed cells, which is feasible with the SNAP‐tag. These applications will be used to demonstrate the potential of self‐labeling protein tags in cell biology. This work was in part funded by a CTI‐grant from the Swiss government (CTI‐grant 6687).