Evaluating the Delivery of Proteins to the Cytosol of Mammalian Cells.
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.
Department of Systems Immunology and Braunschweig Integrated Centre of Systems Biology, Helmholtz Centre for Infection Research, Inhoffenstr. 7, 38124, Braunschweig, Germany.|Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics, Spielmannstr. 7, 38106, Braunschweig, Germany.
Andrea L J
AL
Marschall
Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Paul-Ehrlich-Str. 42-44, 60596, Frankfurt, Germany.
Congcong
C
Zhang
Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics, Spielmannstr. 7, 38106, Braunschweig, Germany. s.duebel@tu-bs.de.
Stefan
S
Dübel
D000818:Animals; D002460:Cell Line; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D004337:Drug Carriers; D018274:Electroporation / Q000379:methods*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D009218:Myosins / Q000276:immunology / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000378:metabolism*; D016707:Tissue Fixation / Q000379:methods; D014404:Tubulin / Q000276:immunology / Q000378:metabolism
D057846:Cell-Penetrating Peptides; D004337:Drug Carriers; D057127:Single-Chain Antibodies; D014404:Tubulin; C045073:Cre recombinase; D019426:Integrases; D009218:Myosins
Antibody; Cell-penetrating peptide (CPP); Electroporation; Profection; Protein delivery; Protein transduction domain (PTD); Protein transfection; Yumab