• Home
  • Search Results
  • A simple fluorescence labeling method for studies of protein oxidation, protein modification, and proteolysis.

A simple fluorescence labeling method for studies of protein oxidation, protein modification, and proteolysis.

Free radical biology & medicine (2011-10-13)
Andrew M Pickering, Kelvin J A Davies
ABSTRACT

Proteins are sensitive to oxidation, and oxidized proteins are excellent substrates for degradation by proteolytic enzymes such as the proteasome and the mitochondrial Lon protease. Protein labeling is required for studies of protein turnover. Unfortunately, most labeling techniques involve (3)H or (14)C methylation, which is expensive, exposes researchers to radioactivity, generates large amounts of radioactive waste, and allows only single-point assays because samples require acid precipitation. Alternative labeling methods have largely proven unsuitable, either because the probe itself is modified by the oxidant(s) being studied or because the alternative labeling techniques are too complex or too costly for routine use. What is needed is a simple, quick, and cheap labeling technique that uses a non-radioactive marker, binds strongly to proteins, is resistant to oxidative modification, and emits a strong signal. We have devised a new reductive method for labeling free carboxyl groups of proteins with the small fluorophore 7-amino-4-methycoumarin (AMC). When bound to target proteins, AMC fluoresces very weakly but when AMC is released by proteinases, proteases, or peptidases, it fluoresces strongly. Thus, without acid precipitation, the proteolysis of any target protein can be studied continuously, in multiwell plates. In direct comparisons, (3)H-labeled proteins and AMC-labeled proteins exhibited essentially identical degradation patterns during incubation with trypsin, cell extracts, and purified proteasome. AMC-labeled proteins are well suited to studying increased proteolytic susceptibility after protein modification, because the AMC-protein bond is resistant to oxidizing agents such as hydrogen peroxide and peroxynitrite and is stable over time and to extremes of pH, temperature (even boiling), freeze-thaw, mercaptoethanol, and methanol.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium cyanoborohydride, reagent grade, 95%
Sigma-Aldrich
7-Amino-4-methylcoumarin, 99%
Sigma-Aldrich
7-Amino-4-methylcoumarin, Chromophore for substrates
Sigma-Aldrich
Sodium cyanoborohydride solution, 5.0 M in 1 M NaOH
Sigma-Aldrich
Sodium cyanoborohydride solution, 1.0 M in THF

Social Media

LinkedIn icon
Twitter icon
Facebook Icon
Instagram Icon

MilliporeSigma

Research. Development. Production.

We are a leading supplier to the global Life Science industry with solutions and services for research, biotechnology development and production, and pharmaceutical drug therapy development and production.

© 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

Reproduction of any materials from the site is strictly forbidden without permission.