Abstract:
:Spectral variants of the green fluorescent protein (GFP) have been extensively used as reporters to image molecular interactions in living cells by fluorescence resonance energy transfer (FRET). However, those GFP variants which are the most efficient donor acceptor pairs for FRET measurements show a high degree of spectral overlap which has hampered in the past their use in FRET applications. Here we use spectral imaging and subsequent un-mixing to quantitatively separate highly overlapping donor and acceptor emissions in FRET measurements. We demonstrate the method in fixed and living cells using a novel GFP based FRET pair (GFP2-YFP (yellow)), which has an increased FRET efficiency compared to the most commonly used FRET pair consisting of cyan fluorescent protein and YFP. Moreover, GFP2 has its excitation maximum at 396 nm at which the YFP acceptor is excited only below the detection level and thus this FRET pair is ideal for applications involving sensitized emission.
journal_name
FEBS Lettjournal_title
FEBS lettersauthors
Zimmermann T,Rietdorf J,Girod A,Georget V,Pepperkok Rdoi
10.1016/s0014-5793(02)03508-1keywords:
subject
Has Abstractpub_date
2002-11-06 00:00:00pages
245-9issue
2eissn
0014-5793issn
1873-3468pii
S0014579302035081journal_volume
531pub_type
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