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The split luciferase complementation assay.

Authors
  • Kato, Naohiro1
  • Jones, Jason
  • 1 Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, USA.
Type
Published Article
Journal
Methods in molecular biology (Clifton, N.J.)
Publication Date
Jan 01, 2010
Volume
655
Pages
359–376
Identifiers
DOI: 10.1007/978-1-60761-765-5_24
PMID: 20734273
Source
Medline
License
Unknown

Abstract

A split luciferase complementation assay to study protein-protein interactions within Arabidopsis protoplasts in 96-well plates is described in this protocol. Two proteins of interest, a bait and prey, which are genetically fused to amino- and carboxy-terminal fragments of Renilla luciferase, are transiently expressed in protoplasts. Physical interactions of these bait and prey proteins reconstitute some of the luciferase activity and result in light emission in the presence of the luciferase substrate. This luminescence is then measured by a microplate luminometer. Amounts of the bait protein accumulated in the protoplasts can be estimated by Western blotting using an antibody that recognizes the amino-terminal fragment of Renilla luciferase. The most advantageous aspect of this assay is its capacity of detecting both association and dissociation of a protein pair of interest in a large-scale format.

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