线粒体蛋白的MitoTracker Green标记，以及通过毛细管电泳和激光诱导的荧光检测进行的后续分析。
Journal of Chromatography B
(
IF
2.8
)
Pub Date : 2003-07-26
, DOI:
10.1016/s1570-0232(03)00371-4
Andrew D. Presley
1
,
Kathryn M. Fuller
2
,
Edgar A. Arriaga
2
Affiliation
Department of Chemistry, University of Minnesota, 207 Pleasant St SE, Minneapolis, MN 55455, USA.
Department of Chemistry University of Minnesota 207 Pleasant St SE Minneapolis MN 55455 USA
MitoTracker Green（MTG）是线粒体选择性荧光标记，通常用于共聚焦显微镜和流式细胞术。预期该染料通过与半胱氨酸残基的游离硫醇基团反应而选择性地累积在线粒体基质中，在此处与线粒体蛋白共价结合。在这里，我们证明MTG可以用作蛋白质标记试剂，与随后通过毛细管电泳和激光诱导的荧光检测（CE-LIF）进行的分析兼容。尽管MTG标记的蛋白和MTG似乎没有电泳分离，但产物荧光强度的增强表明只有具有游离巯基的蛋白才能够与MTG反应。此外，我们建议MTG是对某些线粒体蛋白的部分选择性标记。这种选择性源于线粒体基质中的高MTG浓度，该浓度有利于该亚细胞区室中可用硫醇基团的烷基化。为此，我们处理了通过NS-1细胞裂解液的离心分离而制备的线粒体富集级分。该级分用含有SDS的缓冲液溶解并通过CE-LIF进行分析。具有比单独的MTG强的荧光的条带的存在也表明存在MTG-蛋白质产物。通过用5-呋喃基喹啉-3-羧甲醛（FQ）（与伯氨基反应的荧光试剂）处理溶解的线粒体级分，并通过CE-LIF使用两个独立的检测通道进行分析，从而确定MTG正在标记线粒体蛋白：对于MTG标记的物种，nm为255 nm；对于FQ标记的物种，635 nm。
MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection.
MitoTracker Green (MTG) is a mitochondrial-selective fluorescent label commonly used in confocal microscopy and flow cytometry. It is expected that this dye selectively accumulates in the mitochondrial matrix where it covalently binds to mitochondrial proteins by reacting with free thiol groups of cysteine residues. Here we demonstrate that MTG can be used as a protein labeling reagent that is compatible with a subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Although the MTG-labeled proteins and MTG do not seem to electrophoretically separate, an enhancement in fluorescence intensity of the product indicates that only proteins with free thiol groups are capable of reacting with MTG. In addition we propose that MTG is a partially selective label towards some mitochondrial proteins. This selectivity stems from the high MTG concentration in the mitochondrial matrix that favors alkylation of the available thiol groups in this subcellular compartment. To that effect we treated mitochondria-enriched fractions that had been prepared by differential centrifugation of an NS-1 cell lysate. This fraction was solubilized with an SDS-containing buffer and analyzed by CE-LIF. The presence of a band with fluorescence stronger than MTG alone also indicated the presence of an MTG-protein product. Confirming that MTG is labeling mitochondrial proteins was done by treating the solubilized mitochondrial fraction with 5-furoylquinoline-3-carboxaldehyde (FQ), a fluorogenic reagent that reacts with primary amino groups, and analysis by CE-LIF using two separate detection channels: 520 nm for MTG-labeled species and 635 nm for FQ-labeled species. In addition, these results indicate that MTG labels only a subset of proteins in the mitochondria-enriched fraction.
