Abstract
Many questions concerning the function of the cytoskeleton can be addressed using functional antibodies either in in vitro assays or in whole cell systems. Ultimately, the function of a component has to be studied within intact cell systems. A problem, which one has to be overcome, is the delivery of the antibody into the cells. The delivery of antibodies can be a particular problem either, if large numbers of cells will be analyzed or if one wants to study adhesive as well as non-adhesive cells. Several approaches to introduce proteins and other components into cells have been published, such as electroporation (1), scrape loading (2), or delivery via liposomes (3). Among these the most widespread are microinjection and electroporation. While microinjection is routinely used for adhesive cells, this method will be very time consuming when large numbers of cells have to be studied. Also, it is not feasible to employ this technique for small cells in suspension. Electroporation, on the other hand, has been employed successfully to introduce various components in to cells in suspension. Chang (4,5) has also developed an electroporation method for adhesive cells using an oscillating electric field; still a fare number of cells die during this procedure.
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References
Chakrabarti R., Wylie D. E., and Schuster S. M. (1989) Transfer of monoclonal antibodies into mammalian cells by electroporation. J. Biol. Chem. 264, 15,494–15,500.
McNeil P. L., Murphy R. F., Lanni F., and Taylor D. (1984) A method for incorporating macromolecules into adherent cells. J. Cell Biol. 98, 1556–1564.
Connor J. and Huang L. (1985) Efficient cytoplasmic delivery of a fluorescent dye by pH-sensitive immunoliposomes. J. Cell Biol. 101, 582–589.
Chang D. C. and Reese T. S. (1990) Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy. Biophys. J. 58, 1–12.
Chang D. C. (1997) Experimental strategies in efficient transfection of mamma-lian cells. Electroporation. Meth. Mol. Biol. 62, 307–318.
Fawell S., Seery J., Daikh Y., Moore C., Chen L. L., Pepinsky B., and Barsoum J. (1994) Tat-mediated delivery of heterologous proteins into cells. Proc. Natl. Acad. Sci. USA 91, 664–668.
Frankel A. D. and Pabo C. O. (1988) Cellular uptake of the tat protein from human immunodeficiency virus. Cell 23, 1189–1193.
Vives E., Brodin P., and Lebleun B. (1997) A truncated HIV-1 tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 272, 16,010–16,017.
Steffen W., Karki S., Vaughan K. T., Vallee R. B., Holzbaur L. E., Weiss D. G., and Kuznetsov S. A. (1997) The 74-kD intermediate chain of cytoplasmic dynein is involved in binding the motor to membranous organelles. Molec. Biol. Cell 8, 2077–2088.
Kirsch T., Boehm M., Metzger A. U., Willbold D., Frank R. W., and Rosch P. (1996) Cloning, high-yield expression in Escherichia coli, and purification of biologically active HIV-1 Tat protein. Protein Expr. Purif. 8, 75–84.
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© 2001 Humana Press Inc.
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Steffen, W. (2001). Tat-Mediated Delivery of Antibodies into Cultured Cells. In: Gavin, R.H. (eds) Cytoskeleton Methods and Protocols. Methods in Molecular Biology™, vol 161. Humana Press. https://doi.org/10.1385/1-59259-051-9:141
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DOI: https://doi.org/10.1385/1-59259-051-9:141
Publisher Name: Humana Press
Print ISBN: 978-0-89603-771-7
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