Use of a Small Reporter Molecule To Determine Cell-Surface Proteins by Capillary Electrophoresis and Laser-Induced Fluorescence: Use of 5-SAENTA-x8f for Quantitation of the Human Equilibrative Nucleoside Transporter 1 Protein
| Autor: | Lillian Cook, John R. Mackey, Cheryl Santos, Norman J. Dovichi, Carol E. Cass, Nan Li |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Adenosine
Lysis Population Fluorescence spectrometry Equilibrative nucleoside transporter 1 Fluorescence Analytical Chemistry Flow cytometry Equilibrative Nucleoside Transporter 1 Capillary electrophoresis Tumor Cells Cultured medicine Humans education education.field_of_study Thionucleosides biology medicine.diagnostic_test Chemistry Lasers Electrophoresis Capillary Membrane Proteins Membrane Transport Proteins Affinity Labels Flow Cytometry Molecular biology Biochemistry Cell culture biology.protein Indicators and Reagents Nucleoside |
| Zdroj: | Analytical Chemistry. 74:2573-2577 |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/ac025559r |
| Popis: | The human equilibrative nucleoside transporter 1 protein (hENT1) is a major mediator of cellular entry of nucleosides and anticancer nucleoside drugs; its assay is important in understanding and diagnosing chemotherapy resistance. Here we present a novel assay for quantifying hENT1 using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). A cellular population is treated with 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine-x8-fluorescein (5-SAENTA-x8f), which binds with high affinity and specificity to the hENT1 protein. The cells are washed to remove excess reagent, lysed, and centrifuged, and the supernatant is analyzed by CE-LIF with the use of an internal standard. Accuracy was evaluated by comparing the capillary electrophoresis results with those obtained by flow cytometry; the results were highly correlated, r = 0.96. The relative standard deviation of the hENT1 assay was 10%, determined from nine independent assays of the same cell line, which is 3 times superior to results obtained in a flow cytometry assay. The detection limit for 5-SAENTA-x8f was 4300 molecules injected into the capillary. |
| Databáze: | OpenAIRE |
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