Nucleophosmin/B23 is a key non-ribosomal nucleolar protein expressing in two major isoforms (B23.1 and B23.2), which probably forms not only monomers, but also rather stable oligomeric complexes. An original method for simultaneous quantitative electroblotting and detection on immobilon membranes the both, i.e., oligomeric (190-240 kDa) and monomeric (37-40 kDa) forms of B23, was elaborated. The protocol included modifications as at stages of preparation of HeLa cell lysates and electrophoresis (comparing with Laemmly method), so of electroblotting and protein detection. It has been shown that the number of B23 bands in the oligomere zone is depending not only on the conditions of electrophoresis and electroblotting, but also on a type of detergent used for sample preparation. In cell lysates treated with ionic detergent SDS three oligomer bands with the electrophoretic mobilities of 210-240 kDa were revealed by immunoblot analysis. The number of the oligomeric bands (190-240 kDa) was increased up to seven, when the cells were treated with a non-ionic detergent NP-40. In the both cases, the monomeric forms of B23 were also resolved. The N-terminal sequence analysis of the protein B23 (before and after deformylation) showed that an N-terminal amino acid residue was blocked (but not by the formyl group). So, isoform identification by N-terminal sequencing was impossible. C-terminal amino acid sequence analysis of proteins included in B23-olygomeric complexes by carboxypeptidases allowed to determine which isoforms included and, for the first time, to prove directly that the B23.2 isoform does exist in HeLa cells at the protein level.
|Number of pages
|Published - Jun 1 2004
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology