A practical approach for intracellular protein delivery
- 255 Downloads
Protein delivery represents a powerful tool for experiments in live cells including studies of protein-protein interactions, protein interference with blocking antibodies, intracellular trafficking and protein or peptide biological functions. Most available reagents dedicated to the protein delivery allow efficient crossing of the plasma membrane. Nevertheless, the major disadvantage for these reagents is a weak release of the delivered protein into the cytoplasm. In this publication we demonstrate efficient protein delivery with a non-peptide based reagent, in human epithelial carcinoma HeLa cells and primary human skin fibroblasts. Using a fluorescent protein in combination with fluorescence microscopy and fluorescence-assisted cell sorting analysis, we show that the delivered protein is indeed released effectively in the cytoplasm, as expected for a dedicated carrier. Furthermore, we present a step-by-step method to optimize conditions for successful intracellular protein delivery.
KeywordsIntracellular release Live cells Non-viral technology Protein delivery Synthetic delivery reagent
Bovine serum albumin
Dynamic light scattering
Fluorescence-assisted cell sorting
Protein transduction domain
We are grateful to Drs C. Muller and J. Peluso for their help in FACS analysis and to Drs J.-P. Behr and J.-F. Williamson for critical reading of the manuscript.
- Campbell PL, McCluskey J, Yeo JP, Toh BH (1995) Electroporation of antibodies into mammalian cells. Methods Mol Biol 48:83–92Google Scholar
- Ho A, Schwarze SR, Mermelstein SJ, Waksman G, Dowdy SF (2001) Synthetic protein transduction domains: enhanced transduction potential in vitro and in vivo. Cancer Res 61:474–477Google Scholar
- Labat-Moleur F, Steffan AM, Brisson C, Perron H, Feugeas O, Furstenberger P, Oberling F, Brambilla E, Behr JP (1996) An electron microscopy study into the mechanism of gene transfer with lipopolyamines. Gene Ther 3:1010–1017Google Scholar
- Raptis LH, Liu SK, Firth KL, Stiles CD, Alberta JA (1995) Electroporation of peptides into adherent cells in situ. Biotechniques 18:104, 106, 108, 110 passimGoogle Scholar
- Tinsley JH, Hawker J, Yuan Y (1998) Efficient protein transfection of cultured coronary venular endothelial cells. Am J Physiol 275:H1873–H1878Google Scholar