Nuclear protein import in a permeabilized cell assay

• Verfasst von: Melchior, Frauke [VerfasserIn]
• Titel: Nuclear protein import in a permeabilized cell assay
• Verf.angabe: Frauke Melchior
• Jahr: 1998
• Umfang: 10 S.
• Fussnoten: Gesehen am 07.09.2023
• Titel Quelle: Enthalten in: Protein targeting protocols
• Ort Quelle: Totowa, NJ : Humana Press, 1998
• Jahr Quelle: 1998
• Band/Heft Quelle: (1998), Seite 265-274
• ISBN Quelle: 978-1-59259-572-3
• DOI: doi:10.1385/0-89603-487-9:265
• Datenträger: Online-Ressource
• Sprache: eng
• Sach-SW: FITC Dextran
• Sach-SW: Import Assay
• Sach-SW: Transport Buffer
• Sach-SW: Transport Ligand
• Sach-SW: Transport Reaction
• K10plus-PPN: 1859066283

Abstract

An in vitro nuclear import assay, using digitonin-permeabilized cells, grown on cover slips, and supplemented with exogenous cytosol and fluorescent-transport ligand, was developed in our laboratory several years ago (1). For a general introduction into this technique and references for alternative assays developed by other laboratories, see refs. 2 and 3. Although the original assay faithfully reproduces transport, quantitation by methods such as densitometry on photographic negatives or ACAS (anchored cell analysis and sorting) interactive laser cytometry is a rather time-consuming process. Another disadvantage of the original assay involving attached cells is that the number of cells per assay cannot easily be controlled. Both problems are circumvented by using a modification of the import assay recently developed by Paschal and Gerace (4), and described in detail below. Here, suspension cells are used as the source for permeable cells, and relative transport rates are determined rapidly by flow cytometry (an ELISA-based quantitative assay that allows determination of molar transport rates is described in ref.5).