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

Graziadei, L., Burfeind, P., Bar-Sagi, D. (April 1991) 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. Anal Biochem, 194 (1). pp. 198-203. ISSN 0003-2697 (Print)0003-2697 (Linking)

URL: http://www.ncbi.nlm.nih.gov/pubmed/1714252
DOI: 10.1016/0003-2697(91)90168-S

Abstract

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.

Item Type: Paper
Uncontrolled Keywords: Animals Cell Separation/*methods Cell Survival Cells, Cultured Dextrans/*diagnostic use Flow Cytometry/*methods Fluorescein-5-isothiocyanate Fluoresceins/*diagnostic use Phycoerythrin/analysis Proteins/*analysis Thiocyanates/*diagnostic use
Subjects: Investigative techniques and equipment > flow cytometry
CSHL Authors:
Communities: CSHL labs
Depositing User: Matt Covey
Date: April 1991
Date Deposited: 22 Dec 2015 20:36
Last Modified: 22 Dec 2015 20:36
Related URLs:
URI: http://repository.cshl.edu/id/eprint/32097

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