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A Biotin Ligase-Based Assay for the Quantification of the Cytosolic Delivery of Therapeutic Proteins

  • Wouter P. R. Verdurmen
  • Marigona Mazlami
  • Andreas Plückthun
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1575)

Abstract

The efficient delivery of external proteins from the external milieu to the cytosol of mammalian cells has great potential for both scientific investigations and future therapies. However, when assessing the cellular uptake of proteins, it is often difficult to distinguish between proteins that are stuck in the endosomes and those that have escaped into the cytosol. Here, we describe a method employing the prokaryotic enzyme biotin ligase that overcomes this problem and which can be employed for a highly sensitive quantification of cytosolic protein delivery.

Key words

Biotin Biotin ligase Cellular internalization Cytosolic protein delivery Streptavidin Western blot Protein engineering 

Notes

Acknowledgments

The research leading to these methods has received support from the EU/EFPIA Innovative Medicines Initiative Joint Undertaking COMPACT grant no 115363. W.P.R.V. was supported by a Rubicon grant (Project number: 825.12.002) from the Netherlands Organisation for Scientific Research and the Forschungskredit from the University of Zurich.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Wouter P. R. Verdurmen
    • 1
  • Marigona Mazlami
    • 1
  • Andreas Plückthun
    • 1
  1. 1.Department of BiochemistryUniversity of ZurichZurichSwitzerland

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