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ReAsH: another viable option for in vivo protein labelling in Dictyostelium
Author(s) -
HWANG R.D.,
CHEN C.C.,
KNECHT D.A.
Publication year - 2009
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.2009.03149.x
Subject(s) - green fluorescent protein , fluorescence , fusion protein , dictyostelium , signal peptide , chemistry , dictyostelium discoideum , microbiology and biotechnology , biochemistry , biology , gene , peptide sequence , recombinant dna , physics , quantum mechanics
Summary Biarsenical‐tetracysteine fluorescent protein tagging has been effectively used in a variety of cell types. It has the advantage of requiring a much smaller peptide alteration to existing proteins than fusion to green fluorescent protein (GFP) or monomeric red fluorescent protein (mRFP). However, there are no reports of the tetracysteine tagging system being used in Dictyostelium . In order to establish this tagging system in Dictyostelium , the filamin gene ( FLN ) was modified to express a C‐terminal tetracysteine sequence and then transfected into cells. After addition of either FlAsH‐EDT 2 or ReAsH‐EDT 2 , the fluorescence intensity of cells increased in a time‐dependent manner and reached a plateau after 3 h of incubation. ReAsH had a much stronger and more specifically localized fluorescent signal compared with FlAsH. After removal of the ReAsH‐EDT 2 reagent, the fluorescence signal remained detectable for at least 24 h. The localization of filamin labelled by ReAsH was similar to that of an FLN‐mRFP fusion protein, but the fluorescence signal from the ReAsH‐labelled protein was stronger. Our findings suggest that the ReAsH‐tetracysteine tagging system can be a useful alternative for in vivo protein tagging in Dictyostelium .