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Methods Cell Biol. 2018;143:147-156. doi: 10.1016/bs.mcb.2017.08.009. Epub 2017 Oct 28.

Measurement of lysyl oxidase activity from small tissue samples and cell cultures.

Author information

1
Boston University Henry M. Goldman School of Dental Medicine, Boston, MA, United States. Electronic address: trackman@bu.edu.
2
Boston University Henry M. Goldman School of Dental Medicine, Boston, MA, United States.

Abstract

Increasing interest in the multifunctional lysyl oxidase family of proteins is evident from the growth in the number of new publications each year. The enzymes have unique properties of strong affinities to extracellular matrix components, relative insolubility in typical buffers, low catalytic rates, and often low abundance. Here we provide detailed protocols to extract and assay lysyl oxidase enzymes from tissue samples, cell culture cell layers, and media. Buffer conditions and procedures are optimized based on the characteristics mentioned above, while avoiding the use of radioactive substrates. Peroxidase/Amplex Red-based coupled reactions have proven to be the most useful in this context under specified conditions, and permit calculation of specific enzyme activities in absolute amounts of nanomoles of product/unit time/mg protein.

KEYWORDS:

Amplex Red; Enzyme activity measurement; Fluorescence; Homovanillic acid; Lysyl oxidase extraction; Lysyl oxidase family; Peroxidase-coupled lysyl oxidase enzyme assay

PMID:
29310775
DOI:
10.1016/bs.mcb.2017.08.009
[Indexed for MEDLINE]

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