Design of a fluorescence-activated cell sorting-based Mammalian protein-protein interaction trap.
Methods Mol Biol
; 263: 293-310, 2004.
Article
en En
| MEDLINE
| ID: mdl-14976373
ABSTRACT
The mammalian protein-protein interaction trap (MAPPIT) is a two-hybrid assay based on insights in type I cytokine signal transduction. Bait and prey polypeptides are tethered to mutant cytokine receptor chimeras which are impaired in signaling. On bait-prey interaction and after ligand stimulation, the JAK-STAT signaling cascade is initiated leading to transcription of a reporter or marker gene under the control of the STAT3-responsive rPAP1 promoter. In addition to a physiologically relevant context for mammalian protein-protein interactions this method provides separation of interactor and effector zones, and can be applied for both analytical and screening purposes. In the protocol described here, a cytokine receptor derived surface tag is used as a selectable marker. After an initial presort step using magnetic-activated cell sorting (MACS), "positive" cells are selected by fluorescence-activated cell sorting (FACS).
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Separación Celular
/
Citometría de Flujo
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2004
Tipo del documento:
Article
País de afiliación:
Bélgica