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J Vis Exp. 2013 Dec 5;(82):50968. doi: 10.3791/50968.

Identifying protein-protein interaction in Drosophila adult heads by Tandem Affinity Purification (TAP).

Author information

1
Neuroscience Center of Excellence, Louisiana State University Health Sciences Center.

Abstract

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast(1) to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

PMID:
24335807
PMCID:
PMC4038007
DOI:
10.3791/50968
[Indexed for MEDLINE]
Free PMC Article

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