Measuring FRET in flow cytometry and microscopy.
Curr Protoc Cytom
; Chapter 12: Unit12.8, 2006 Nov.
Article
em En
| MEDLINE
| ID: mdl-18770834
ABSTRACT
This unit presents protocols describing the measurement of protein associations using FRET as determined by flow and image cytometry. The proteins under investigation can be labeled by fluorescent antibodies or fluorescent protein (FP) variants. The flow cytometry protocols determine FRET based on the measurement of donor quenching, which provides a FRET value on a population basis, or based on the measurement of fluorescence intensities in the donor, FRET, and acceptor channels, which provides cell-by-cell FRET values. An extension of this protocol is based on cell-by-cell correction for autofluorescence and requires the measurement of four fluorescence intensities. The algorithm described can be applied in image cytometric FRET as well. The image protocol determines FRET resolved by donor photobleaching. The authors provide extensive discussion of pitfalls, limitations, and interpretation.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Transferência Ressonante de Energia de Fluorescência
/
Citometria de Fluxo
/
Proteínas Luminescentes
Limite:
Humans
Idioma:
En
Ano de publicação:
2006
Tipo de documento:
Article