Protocol abstract
Nature Protocols 2, - 1528 - 1535 (2007)
Published online: 14 June 2007 | doi:10.1038/nprot.2007.209
Subject Category: Isolation, purification and separation
The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins
Thomas GM Schmidt1 & Arne Skerra2
Abstract
The Strep-tag II is an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin and can be fused to recombinant proteins in various fashions. We describe a protocol that enables quick and mild purification of corresponding Strep-tag II fusion proteins—including their complexes with interacting partners—both from bacterial and eukaryotic cell lysates using affinity chromatography on a matrix carrying an engineered streptavidin (Strep-Tactin), which can be accomplished within 1 h. A high-affinity monoclonal antibody (StrepMAB-Immo) permits stable immobilization of Strep-tag II fusion proteins to solid surfaces, for example, for surface plasmon resonance analysis. Selective and sensitive detection on western blots is achieved with Strep-Tactin/enzyme conjugates or another monoclonal antibody (StrepMAB-Classic). Thus, the Strep-tag II, which is short, biologically inert, proteolytically stable and does not interfere with membrane translocation or protein folding, offers a versatile tool both for the rapid isolation of a functional gene product and for its detection or molecular interaction analysis.
- IBA GmbH, Rudolf-Wissell-Strasse 28, 37079 Göttingen, Germany.
- Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany.
Correspondence to: Arne Skerra2 e-mail: skerra@wzw.tum.de
nature-products
A-Z product listing
- 0.1 M N-hydroxysuccinimide(Biacore)
- 0.4 M 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide/HCl, EDC(Pierce)
- 0.5–2 ml bed volume(Pierce)
- 1 M ethanolamine/HCl(Biacore)
- 1 or 5 ml bed volume(IBA)
- BIACORE X instrument(Biometra)
