体外试验中胞内络氨酸酶活性的可视化

Riddhi Atul Jani; Sudeshna Nag; Subba Rao Gangi Setty

doi:10.21769/BioProtoc.1794

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

Visualization of Intracellular Tyrosinase Activity in vitro

体外试验中胞内络氨酸酶活性的可视化

Riddhi Atul Jani ^*

Sudeshna Nag ^*

Subba Rao Gangi Setty email

(*contributed equally to this work) 发布: 2016年04月20日第6卷第8期 DOI: 10.21769/BioProtoc.1794 浏览次数: 9195

评审: Ralph BottcherHsin-Yi ChangAnonymous reviewer(s)

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Journal of Cell Science, featuring study using the protocol.

Sep 2015

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

96孔板NF-κB/AP-1报告基因检测转染细胞中野生型及致病性ALPK1突变体活性

Tom Snelling

2024年11月20日 799 阅读

野生型和致病突变体ALPK1激酶活性的无细胞放射性磷酸化定量测定

Tom Snelling

2024年11月20日 749 阅读

基于荧光偏振的组织蛋白酶L抑制剂高通量筛选方法

Keyu Guo [...] Shuyi Si

2025年07月20日 850 阅读

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.

Keywords: Tyrosinase (酪氨酸酶)

Melanosome (黑素小体)

Oculocutaneous albinism (眼皮肤白化病)

Copper (铜)

L-DOPA (左旋多巴)

Materials and Reagents

Glass coverslips (diameter-12 mm, No.1) (Polar Industrial Corporation, Blue Star, catalog number: 12mm Circular )
Note: See Recipes for acid wash and sterilization.
Micro slides (L-75 mm x W-25 mm x h-1.35 mm) (Polar Industrial Corporation, Blue Star, catalog number: PIC-1 )
Plastic tissue culture (6 well) plate (Corning, catalog number: 3506 ) and bottle-top vacuum filter (pore size 0.22 μm) (Corning, catalog number: 430015 )
Melanocytes (Immortal wild type mouse melanocytes, melan-Ink4a-Arf-1 from C57BL/6J mice, referred to here as melan-Ink4a) [Resource: The Wellcome Trust Functional Genomics Cell Bank (Sviderskaya et al., 2010)]
Copper(II) sulphate pentahydrate (CuSO₄·5H₂O) (Sigma-Aldrich, catalog number: C7631 )
3, 4-Dihydroxy-D-phenylalanine (D-DOPA) (Sigma-Aldrich, catalog number: D9378 )
3, 4-Dihydroxy-L-phenylalanine (L-DOPA) (Sigma-Aldrich, catalog number: D9628 )
HCl (Sigma-Aldrich, catalog number: H1758 )
HNO₃ (Merck Millipore, catalog number: 101799 )
KCl (Sigma-Aldrich, catalog number: P9541 )
KH₂PO₄ (Merck Millipore, catalog number: 104873 )
NaCl (Fisher Scientific, catalog number: BP358-1 )
Na₂HPO₄·2H₂O (Fisher Scientific, catalog number: S472-500 )
Ethanol (70%) (Merck Millipore, catalog number: 818760 )
Fetal bovine serum (Biowest, catalog number: S1810-500 )
Formaldehyde (36.5-38% in H₂O) solution (HCHO solution) (Sigma-Aldrich, catalog number: F8775 )
Fluromount-G or mounting medium (SouthernBiotech, catalog number: 0100-01 )
Matrigel Matrix (Corning Matrigel Growth Factor Reduced Basement Membrane Matrix, Phenol Red-Free) (Corning, catalog number: 356231 )
Penicillin-Streptomycin (antibiotic) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140-122 )
RPMI-1640 media (Thermo Fisher Scientific, Gibco^TM, catalog number: 31800-022 )
L-Glutamine (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030-081 )
0.1% DOPA solution (see Recipes)
4% Formaldehyde solution (see Recipes)
Growth media (see Recipes)
0.1% Matrigel matrix solution (see Recipes)
1x PBS (see Recipes)

Equipment

CO₂ incubator (maintained at 37 °C, 10% CO₂) (Thermo Fisher Scientific, model: Forma Water Jacketed CO₂ incubator )
Forceps (sterilized by autoclave)
Bright field microscope (Olympus Corporation, model: IX81 motorized inverted fluorescence microscope )
Hot-air-oven (Eyela, catalog number: NDO-420W )
Glass beaker (250 ml) (Borosil, catalog number: 1000D21 )

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Jani, R. A., Nag, S. and Setty, S. R. G. (2016). Visualization of Intracellular Tyrosinase Activity in vitro. Bio-protocol 6(8): e1794. DOI: 10.21769/BioProtoc.1794.