The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications

Research output: Contribution to journalArticle


Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a rationally designed epitope tag that serves a remarkably broad spectrum of applications in life sciences while outperforming established tags like the HA-, FLAG®- or myc-tag. The ALFA-tag forms a small and stable α-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We characterize a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in vivo detection of proteins. We show the crystal structure of the complex that enabled us to design a nanobody mutant (NbALFAPE) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.


  • Hansjörg Götzke
  • Markus Kilisch
  • Markel Martínez-Carranza
  • Shama Sograte-Idrissi
  • Abirami Rajavel
  • Thomas Schlichthaerle
  • Niklas Engels
  • Ralf Jungmann
  • Pål Stenmark
  • Felipe Opazo
  • Steffen Frey
External organisations
  • Stockholm University
  • NanoTag Biotechnologies GmbH
  • University Medical Center Göttingen
  • Max Planck Institute of Biochemistry
  • Ludwig-Maximilian University of Munich
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Medical Biotechnology
Original languageEnglish
Article number4403
JournalNature Communications
Publication statusPublished - 2019 Sep 27
Publication categoryResearch