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An optimized protocol for protein purification in cultured mammalian cells using a tandem affinity purification approach

Abstract

This protocol describes a method that we developed to adapt the tandem affinity purification (TAP) approach for use in mammalian cells. The protocol involves fusing a protein of interest with a tandem tag consisting of two FLAG tags (FF) followed by two protein-A immunoglobulin G (IgG) binding domains (ZZ). The protocol improves upon previously published TAP approaches by employing FLAG in place of calmodulin binding peptide (CBP) with resulting higher recovery during purification. In addition, we use a bicistronic expression system that ensures recovery of stably transfected cell lines expressing easily detectable levels of the protein of interest. A method is also presented for generating cytoplasmic and nuclear extracts, which extends use of this protocol to identify protein–protein interactions occurring specifically in the cytoplasm or nucleus. This protocol facilitates the preparation of partially purified recombinant protein and identification of protein–protein interactions in mammalian cell culture models. The protocol can be completed in 34 h.

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Figure 1: Schematic of the expression vector and tandem epitope tag and positions and sequence of usable restriction sites for cloning.
Figure 2: FLAG Western blot analysis of samples collected at sequential steps during tandem affinity purification (TAP) of nuclear extract from 293T cells stably transfected with pIRES-puro3Δint-WT1-cFF-ZZ and selected using medium containing 3 μg ml−1 puromycin.
Figure 3: Silver stain analysis of samples collected at sequential steps during tandem affinity purification (TAP) of nuclear extract from 293T cells stably transfected with pIRES-puro3Δint-WT1-cFF-ZZ.

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Correspondence to Russ P Carstens.

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Tsai, A., Carstens, R. An optimized protocol for protein purification in cultured mammalian cells using a tandem affinity purification approach. Nat Protoc 1, 2820–2827 (2006). https://doi.org/10.1038/nprot.2006.371

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