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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 Riddhi Atul Jani *
Sudeshna Nag Sudeshna Nag *
Subba Rao Gangi Setty Subba Rao Gangi Setty

 (*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: 9822

评审: Ralph BottcherHsin-Yi ChangAnonymous reviewer(s)

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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) search Melanosome (黑素小体) search Oculocutaneous albinism (眼皮肤白化病) search Copper (铜) search L-DOPA (左旋多巴) search

Materials and Reagents

  1. Glass coverslips (diameter-12 mm, No.1) (Polar Industrial Corporation, Blue Star, catalog number: 12mm Circular )
    Note: See Recipes for acid wash and sterilization.
  2. Micro slides (L-75 mm x W-25 mm x h-1.35 mm) (Polar Industrial Corporation, Blue Star, catalog number: PIC-1 )
  3. Plastic tissue culture (6 well) plate (Corning, catalog number: 3506 ) and bottle-top vacuum filter (pore size 0.22 μm) (Corning, catalog number: 430015 )
  4. 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)]
  5. Copper(II) sulphate pentahydrate (CuSO4·5H2O) (Sigma-Aldrich, catalog number: C7631 )
  6. 3, 4-Dihydroxy-D-phenylalanine (D-DOPA) (Sigma-Aldrich, catalog number: D9378 )
  7. 3, 4-Dihydroxy-L-phenylalanine (L-DOPA) (Sigma-Aldrich, catalog number: D9628 )
  8. HCl (Sigma-Aldrich, catalog number: H1758 )
  9. HNO3 (Merck Millipore, catalog number: 101799 )
  10. KCl (Sigma-Aldrich, catalog number: P9541 )
  11. KH2PO4 (Merck Millipore, catalog number: 104873 )
  12. NaCl (Fisher Scientific, catalog number: BP358-1 )
  13. Na2HPO4·2H2O (Fisher Scientific, catalog number: S472-500 )
  14. Ethanol (70%) (Merck Millipore, catalog number: 818760 )
  15. Fetal bovine serum (Biowest, catalog number: S1810-500 )
  16. Formaldehyde (36.5-38% in H2O) solution (HCHO solution) (Sigma-Aldrich, catalog number: F8775 )
  17. Fluromount-G or mounting medium (SouthernBiotech, catalog number: 0100-01 )
  18. Matrigel Matrix (Corning Matrigel Growth Factor Reduced Basement Membrane Matrix, Phenol Red-Free) (Corning, catalog number: 356231 )
  19. Penicillin-Streptomycin (antibiotic) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  20. RPMI-1640 media (Thermo Fisher Scientific, GibcoTM, catalog number: 31800-022 )
  21. L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030-081 )
  22. 0.1% DOPA solution (see Recipes)
  23. 4% Formaldehyde solution (see Recipes)
  24. Growth media (see Recipes)
  25. 0.1% Matrigel matrix solution (see Recipes)
  26. 1x PBS (see Recipes)

Equipment

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

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

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.

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分类

生物化学 > 蛋白质 > 活性

细胞生物学 > 细胞染色 > 蛋白质

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