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
| Autor: | Patricia Burfeind, Dafna Bar-Sagi, Linda Graziadei |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
Cell Survival
Population Cell Biophysics Cell Separation Biology Biochemistry Flow cytometry chemistry.chemical_compound medicine Animals Fluorescein education Fluorescein isothiocyanate Molecular Biology Cells Cultured education.field_of_study medicine.diagnostic_test Electroporation Proteins Dextrans Phycoerythrin Cell Biology Cell sorting Flow Cytometry Fluoresceins medicine.anatomical_structure Dextran chemistry Fluorescein-5-isothiocyanate Thiocyanates |
| Zdroj: | Analytical Biochemistry. 194:198-203 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/0003-2697(91)90168-s |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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