Isolation of ubiquitin-E2 (ubiquitin-conjugating enzyme) complexes from erythroleukaemia cells using immunoaffinity techniques

The Biochemical Journal
K TakadaK Ohkawa

Abstract

A variety of ubiquitin-associated (or conjugated) proteins, including substrates and enzymes for the ubiquitin system, are present in eukaryotic cells. In the present study we developed a simple method for their isolation, consisting of immunoaffinity chromatography using the monoclonal antibody FK2, which recognizes the conjugated ubiquitin molecule. Using this method followed by gel filtration, we isolated multi-ubiquitinated proteins with high molecular masses (>30 kDa) and also ubiquitinthioester-linked and mono-ubiquitinated forms of ubiquitin-conjugating (E2) enzymes, UbcH7 and UBE2N, together with mono-, di- and tri-ubiquitin molecules, from the cytoplasmic extract of heat-shock-treated K562 erythroleukaemia cells. We also demonstrated that the FK2 antibody was capable of precipitating a ubiquitin-UbcH7 thioester, but not free UbcH7, which enabled the measurement of the respective cellular levels separately. The immunoprecipitable ubiquitin-UbcH7 thioester was found only when the cells were treated with heat-shock. These results suggest the usefulness of the immunoaffinity techniques for identifying and analysing the cellular enzyme/protein-ubiquitin complexes.

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Citations

Nov 30, 2002·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Thomas C HunterPaul A Haynes
Sep 29, 2009·Protein Science : a Publication of the Protein Society·Jing SongYuan Chen
Feb 27, 2014·Biotechnology & Genetic Engineering Reviews·Guoqiang Xu, Samie R Jaffrey
Jun 15, 2014·Journal of Cell Science·Fabienne C FieselWolfdieter Springer

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