title: Tandem Affinity Purification in Drosophila Heads and Ovaries.
abstract: Tandem affinity purification (TAP) (Pugiet al., 2001; Rigautet al., 1999) is a method that uses a tagging approach of a target protein of interest for a two-step purification scheme in order to pull down protein complexes under native conditions and expression levels. The TAP tag consists of three components: a calmodulin-binding peptide, aTobacco etch virus(TEV) protease cleavage site and Protein A which is an immunoglobulin G (IgG)-binding domain. This protocol was modified from the original methodology used in yeast cells (Pugiet al., 2001; Rigautet al., 1999) for isolation of protein complexes from Drosophila heads and ovaries expressing a TAP tagged protein of interest. To determine in vivo binding partners of the Drosophila fragile X protein (dFMR1), we developed a transgenic strain of flies expressing a recombinant form of dFMR1 with a carboxy-terminal TAP tag (Tsai and Carstens, 2006). To ensure that the construct was expressed at wild-type levels, we engineered this form of the tagged protein in the context of a genomic rescue construct that rescued a mutant sterility phenotype. The purification process was performed using mild conditions to maintain native protein interactions. For TAP methods in Drosophila S2 cell culture, we have successfully used a protocol previously published by Tsai and Carstens (Tsai and Carstens, 2006; Bhogalet al., 2011).
authors.affiliation: Department of Genetics, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA.
authors.forename: Anita
authors.initials: A
authors.lastname: Pepper
authors.affiliation: Department of Genetics, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA.
authors.forename: Balpreet
authors.initials: B
authors.lastname: Bhogal
authors.affiliation: Department of Genetics, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Jongens