Immunoassay for the quantification of intracellular multi-ubiquitin chains.
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.
Takada
K
K
Department of Biochemistry, Jikei University School of Medicine, Tokyo, Japan.
Nasu
H
H
Hibi
N
N
Tsukada
Y
Y
Ohkawa
K
K
Fujimuro
M
M
Sawada
H
H
Yokosawa
H
H
D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D005069:Evaluation Studies as Topic; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D011487:Protein Conformation; D011817:Rabbits; D012015:Reference Standards; D012156:Reticulocytes / Q000737:chemistry; D012680:Sensitivity and Specificity; D014452:Ubiquitins / Q000032:analysis* / Q000737:chemistry / Q000592:standards
D014452:Ubiquitins