Ability of ubiquitin radioimmunoassay to discriminate between monoubiquitin and multi-ubiquitin chains.
Free ubiquitin (mainly monoubiquitin) and multi-ubiquitin chains coexist in eukaryote cells and serve distinct cellular roles. However, any immunoassay systems established previously have not been proved to be applicable for measuring the former without cross-reactive responses with the latter. For this purpose, we developed a radioimmunoassay specific to monoubiquitin by employing antiserum US-1 against ubiquitin. In this assay, ubiquitin-protein conjugates, prepared by a reticulocyte lysate fraction II and fractionated on Moro Q and Superdex 200 columns, exhibited practically no cross-reactivity. The cross-reactivity of fractionated ubiquitin-lysozyme conjugates was also analyzed as a function of their multi-ubiquitin chain size. As a result, the larger the conjugates were found to be, the weaker were the cross-reactive responses they showed, and the multi-ubiquitin chains (n > approx. 20) were substantially unreactive in the radioimmunoassay. By using the radioimmunoassay, heat-shock-induced decrease in the level of cellular free (mono)ubiquitin was detected. In addition, the standard preparation of multi-ubiquitin chains was not cross-reactive in all other five radioimmunoassays employing distinct antibodies to ubiquitin (four antisera and a monoclonal antibody). These data suggest that radioimmunoassays employing ubiquitin antibodies raised by the general methods can discriminate between monoubiquitin and multi-ubiquitin chains and quantitate cellular free ubiquitin.
Takada
K
K
Department of Biochemistry I, Jikei University School of Medicine, Tokyo, Japan. MHA03643@niftyserve.or.jp
Hibi
N
N
Tsukada
Y
Y
Shibasaki
T
T
Ohkawa
K
K
D000818:Animals; D001704:Biopolymers / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D025821:Polyubiquitin; D011863:Radioimmunoassay / Q000379:methods*; D051381:Rats; D012156:Reticulocytes / Q000737:chemistry; D012680:Sensitivity and Specificity; D013347:Subcellular Fractions / Q000737:chemistry; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000032:analysis*
D001704:Biopolymers; D014452:Ubiquitins; D025821:Polyubiquitin