Evaluating the Delivery of Proteins to the Cytosol of Mammalian Cells

  • Andrea L. J. Marschall
  • Congcong Zhang
  • Stefan DübelEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1513)


Delivery of proteins to the cytosol of living cells is a promising research tool. Delivery of antibodies in particular bears exciting applications such as in vivo tracking of proteins at endogenous expression levels or interference with cellular processes. In spite of the large number of methods published for protein delivery, successful applications so far are rare. A possible explanation for this is a vast overestimation of the delivery efficiency due to the use of inappropriate detection methods and/or unsuitable positive controls for cytosolic delivery. Therefore, we provide strategies for unequivocally detecting cytoplasmic protein delivery and quantifying protein transformation efficiency. Finally, we present a protocol for efficient protein delivery to the cytosol validated using these methods.

Key words

Antibody Profection Protein transfection Protein delivery Cell-penetrating peptide (CPP) Protein transduction domain (PTD) Electroporation Yumab 



We are grateful to André Frenzel and Thomas Schirrmann for helpful discussions and the EU consortium “Affinomics” for financial support.


  1. 1.
    Marschall AL, Frenzel A, Schirrmann T et al (2011) Targeting antibodies to the cytoplasm. MAbs 3:3–16CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Marschall AL, Dübel S, Böldicke T (2015) Specific in vivo knockdown of protein function by intrabodies. MAbs 7:1010–1035CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Richard JP, Melikov K, Vives E et al (2003) Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J Biol Chem 278:585–590CrossRefPubMedGoogle Scholar
  4. 4.
    Marschall AL, Zhang C, Frenzel A et al (2014) Delivery of antibodies to the cytosol: debunking the myths. MAbs 6:943–956CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Will E, Klump H, Heffner N et al (2002) Unmodified Cre recombinase crosses the membrane. Nucleic Acids Res 30:e59CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Moutel S, El Marjou A, Vielemeyer O et al (2009) A multi-Fc-species system for recombinant antibody production. BMC Biotechnol 9:14CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Schirrmann T, Büssow K (2010) Transient production of scFv-Fc fusion proteins in mammalian cells. In: Stefan Dübel RK (ed) Antibody engineering. Springer, New York, pp 387–398CrossRefGoogle Scholar
  8. 8.
    Jäger V, Büssow K, Wagner A et al (2013) High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells. BMC Biotechnol 13:52CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Clarke M, Spudich JA (1977) Nonmuscle contractile proteins: the role of actin and myosin in cell motility and shape determination. Annu Rev Biochem 46:797–822CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Andrea L. J. Marschall
    • 1
    • 2
  • Congcong Zhang
    • 3
  • Stefan Dübel
    • 2
    Email author
  1. 1.Department of Systems Immunology and Braunschweig Integrated Centre of Systems BiologyHelmholtz Centre for Infection ResearchBraunschweigGermany
  2. 2.Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and BioinformaticsBraunschweigGermany
  3. 3.Georg-Speyer-Haus, Institute for Tumor Biology and Experimental TherapyFrankfurtGermany

Personalised recommendations