Introduction of unlabeled proteins into living cells by electroporation and isolation of viable protein-loaded cells using dextran-fluorescein isothiocyanate as a marker for protein uptake.
Commonly, microinjection has been the method of choice for introducing proteins into living cells. Viable cells containing an introduced protein can be then identified providing that the protein is fluorochrome conjugated. This approach is applicable only for adherent cells, and the number of cells that can be analyzed is small. In this study, we have established that electroporation can be used to load proteins into large numbers of cells with high efficiency. Furthermore, we have developed a method for the isolation of protein-loaded cells using fluorescein isothiocyanate-dextran (dextran-FITC) as a molecular marker for protein uptake. The essential features of this method are that dextran-FITC is included in the electroporation medium and, thus, is cointroduced with the protein of interest. Purification of cells containing dextran-FITC using fluorescence-activated cell sorting yields a population which is composed almost entirely of cells containing the protein of interest.
Graziadei
L
L
Cold Spring Harbor Laboratory, New York 11724.
Burfeind
P
P
Bar-Sagi
D
D
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D003911:Dextrans; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D010799:Phycoerythrin / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D013861:Thiocyanates
D003911:Dextrans; D005452:Fluoresceins; D011506:Proteins; D013861:Thiocyanates; D010799:Phycoerythrin; D016650:Fluorescein-5-isothiocyanate