| Visualization of Intracellular Tyrosinase Activity in vitro
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Author:
Date:
2016-04-20
[Abstract] Melanocytes produce the melanin pigments in melanosomes and these organelles protect the skin against harmful ultraviolet rays. Tyrosinase is the key cuproenzyme which initiates the pigment synthesis using its substrate amino acid tyrosine or L-DOPA (L-3, 4-dihydroxyphenylalanine). Moreover, the activity of tyrosinase directly correlates to the cellular pigmentation. Defects in tyrosinase transport to melanosomes or mutations in the enzyme or reduced intracellular copper levels result in loss of tyrosinase activity in melanosomes, commonly observed in albinism. Here, we describe a method to detect the intracellular activity of tyrosinase in mouse melanocytes. This protocol will visualize the active tyrosinase present in the intracellular vesicles or organelles including melanosomes.
[摘要] 黑素细胞在黑素体中产生黑色素，这些细胞器保护皮肤免受有害的紫外线。 酪氨酸酶是使用其底物氨基酸酪氨酸或L-DOPA（L-3,4-二羟基苯丙氨酸）引发颜料合成的关键铜酶蛋白酶。 此外，酪氨酸酶的活性与细胞色素沉着直接相关。 酪氨酸酶转运到黑素体中的缺陷或酶中的突变或降低的细胞内铜水平导致黑素体中酪氨酸酶活性的丧失，通常在白化病中观察到。 在这里，我们描述了一种方法来检测小鼠黑素细胞中酪氨酸酶的细胞内活性。 该协议将使存在于细胞内囊泡或细胞器（包括黑素体）中的活性酪氨酸酶可视化。
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| Dictyostelium Cultivation, Transfection, Microscopy and Fractionation
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Author:
Date:
2015-06-05
[Abstract] The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, chemotaxis, cell division and membrane trafficking. In addition the fractionation of cells and isolation of organelles or known compartments can often verify any subcellular localisation and the use of tagged proteins as bait for the immunoprecipitation of material from cell fractions can identify specific binding partners and multiprotein complexes thereby helping assign a function to the tagged protein. We have successfully applied these techniques to the Dictyostelium discoideum protein TSPOON that is part of an ancient ...
[摘要] 使用更复杂的显微镜，活细胞中荧光标记的蛋白质的实时可视化大大增加了我们对基本生理过程如细胞运动，趋化性，细胞分裂和膜运输过程中关键蛋白质动力学的了解。此外，细胞的分级和分离细胞器或已知的隔室通常可以验证任何亚细胞定位，并且使用标记的蛋白质作为诱饵用于来自细胞部分的物质的免疫沉淀可以鉴定特异性结合配偶体和多蛋白复合物，从而有助于赋予功能标记蛋白。我们已经成功地将这些技术应用于作为古代异构六聚体膜转运复合物的一部分的盘基网柄菌discoideum蛋白TSPOON（Hirst等，2013）。 ...
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| Hypertrophy Analysis and Quantification in Embryonic Cardiomyocites
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Author:
Date:
2013-10-20
[Abstract] Myocardial growth goes from proliferation to hypertrophy during development. The measurement of the relative cell area provides information of cardiomyocyte hypertrophy, which is ideal for studying myocardial development.
[摘要] 心肌生长在发育期间从增殖到肥大。 相对细胞面积的测量提供心肌细胞肥大的信息，这是研究心肌发育的理想选择。
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