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 (> 30kDa) 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.
- affinity chromatography
Abbreviations used: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; MUCRP1, multi-ubiquitin chain reference preparation 1; DTT, dithiothreitol; MALDI–TOF-MS, matrix-assisted laser-desorption ionization–time-of-flight MS.
- The Biochemical Society, London ©2001