Protocol

Bimolecular Affinity Purification (BAP): Tandem Affinity Purification Using Two Protein Baits

¹ Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, MI 48073, USA
² Department of Biochemistry and Molecular Biology, School of Medicine, Hebrew University in Jerusalem, Jerusalem 91120, Israel
³ Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA
⁴ Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA

⁵ These authors contributed equally to the work.

⁶Corresponding author (Ezra.Burstein{at}UTSouthwestern.edu).

INTRODUCTION

The tandem affinity purification (TAP) procedure was pioneered in yeast for the purpose of purifying and characterizing protein complexes. While affinity purification is relatively easy to perform, nonspecific protein interactions can plague the identification of true interacting partners of the given bait utilized in the purification. To alleviate this problem, two sequential affinity purification steps are employed in the TAP procedure. Since its inception in yeast, TAP has gone through many adaptations and has been employed multiple times in diverse organisms, including mammalian systems. In all these approaches, two out of many possible affinity moieties are employed and are usually expressed as a fusion polypeptide in the amino or carboxyl-terminal region of the protein bait. In this protocol, we describe a variation on the TAP procedure in which the affinity moieties are placed on two different proteins of a molecular complex to isolate or detect components present in the complex. This variation, which we refer to as bimolecular affinity purification (BAP), is suited for the identification of specific molecular complexes marked by the presence of two known components.