Immunoassay for the quantification of intracellular multi-ubiquitin chains

Eur J Biochem. 1995 Oct 1;233(1):42-7. doi: 10.1111/j.1432-1033.1995.042_1.x.

Abstract

A sandwich ELISA has been developed to measure intracellular levels of multi-ubiquitin chains. The mixture of multi-ubiquitin chains, prepared in vitro by incubation of ubiquitin (plus 125I-ubiquitin) and lysozyme with ubiquitin-ligating enzymes and ATP, was partially purified and established as a standard named the multi-ubiquitin-chain reference preparation 1 (MUCRP1). The concentration of MUCRP1 was calculated from the recovered radioactivity of 125I-ubiquitin. All measurements by the ELISA were expressed in terms of MUCRP1. The ELISA showed good sensitivity (98 pg/ml), precision (intra-assays < 6%) and reproducibility (interassay < 9%). In addition, there was no substantial cross-reaction with mono-, di- and tri-ubiquitin, or mono-ubiquitinated and di-ubiquitinated lysozyme in the ELISA, and large multi-ubiquitin chains (n > approximately 6) may be fully reactive. These results combined with excellent results in the recovery and dilution tests guarantee accurate measurement of multi-ubiquitin chains in cell extracts prepared with a lysis buffer (water soluble) or the buffer supplemented 8 M urea (urea soluble). The level of the water-soluble multi-ubiquitin chains in reticulocytes was lower than that of erythrocytes, but the urea-soluble chain level was higher in the reticulocytes. Heat-shock treatment of HeLa cells increased the urea-soluble multi-ubiquitin chains. These data indicate that this ELISA provides a useful and reliable approach to the study of intracellular multi-ubiquitin-conjugate turnover.

MeSH terms

  • Animals
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Enzyme-Linked Immunosorbent Assay / standards
  • Enzyme-Linked Immunosorbent Assay / statistics & numerical data
  • Evaluation Studies as Topic
  • HeLa Cells
  • Humans
  • Mice
  • Protein Conformation
  • Rabbits
  • Reference Standards
  • Reticulocytes / chemistry
  • Sensitivity and Specificity
  • Ubiquitins / analysis*
  • Ubiquitins / chemistry
  • Ubiquitins / standards

Substances

  • Ubiquitins