Electrophoretic analysis of HeLa cell and human liver nucleolar proteins and antigens.
The major antigens in HeLa nucleolar extracts recognized by immunoblots with rabbit antisera had molecular weights of 145, 110, and 34 kd. The major antigens in the HeLa nucleolar residues had molecular weights of 145, 110, 86, 68, 55, 48, and 34 kd. In the liver, the major antigens in the nucleolar extracts had molecular weights of 86 and 76 kd. In the liver residues, the major antigens had molecular weights of 110, 86, 76, 65, and 55 kd. On two-dimensional gels stained with Coomassie blue, the HeLa nucleolar extract contained large amounts of protein B23 and lesser amounts of protein C23. In the liver nucleolar samples separated on two-dimensional (2-D) gels, protein 55/7.6 (Mr/pI) was the major protein in the extract. Lesser amounts of protein B23 were identified. Addition of protease inhibitors markedly improved the quality of the protein samples as shown in one-dimensional gel patterns for liver nucleolar proteins and to a lesser extent for HeLa nucleolar proteins. In the 2-D immunoblots of the HeLa extract and HeLa residues, the major stained band was protein C23 (110/5.2-5.6). In the liver extract, the major bands were 70/5.8-7 and 60/5.8-7. With a monoclonal antibody (MS-3) to protein C23 a 76/5.8 band was more notable in the residue which supported the results of the Coomassie blue and immunostains with rabbit antinucleolar antibodies. Some degradation products of protein C23 were observed in both the liver extract and the liver residue despite the use of protease inhibitors. Protein C23 along with other proteins are major antigens in HeLa nucleoli. In human liver nucleoli, a major protein 55/7.6 was identified which was not observed in the HeLa extracts. These studies show that a combination of protease inhibitors markedly reduced degradation of proteins in liver samples and provided a more satisfactory sample for comparison with HeLa cells. Qualitative and quantitative differences were found in the nucleolar proteins by Coomassie blue and immunostaining.
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D000941:Antigens / Q000032:analysis*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000032:analysis; D006801:Humans; D008099:Liver / Q000032:analysis*; D009687:Nuclear Proteins; D000090243:Nucleophosmin; D012261:Ribonucleoproteins / Q000032:analysis*; D012394:Rosaniline Dyes; D013194:Staining and Labeling
D000941:Antigens; D009687:Nuclear Proteins; D012261:Ribonucleoproteins; D012394:Rosaniline Dyes; D000090243:Nucleophosmin; C004692:coomassie Brilliant Blue; D004229:Dithiothreitol