Purification of an 86-70 kDa nuclear DNA-associated protein complex.
In the course of studies on nucleolar antigens, monoclonal antibodies were developed, one of which recognized an 86 kDa antigen as shown by analysis of nuclear extracts from HeLa or Namalwa cells. Immunofluorescence studies on HeLa cells showed a nucleoplasmic and phase-dependent nucleolar localization of the monoclonal antibody was decreased after digestion with DNAase I but not with RNAase A. For purification, the antigen was released from nuclei by digestion with DNAase I and then purified by chromatography on DEAE cellulose, phosphocellulose and antibody-Sepharose affinity chromatography. Interestingly, the immunoaffinity purified product contained two polypeptide chains; the immunoreactive polypeptide had an Mr of 86 000 and a pI of 6.0. The complex also contained a 70 kDa, pI 6.5 nonantigenic polypeptide in a 1:1 ratio. The overall purification of the complex was 5700-fold. Both polypeptides contained approx. 15 mol% glutamic acid and the 70 kDa polypeptide contained approx. 15 mol% serine.
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Yaneva
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Ochs
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McRorie
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Zweig
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Busch
D000596:Amino Acids / Q000032:analysis; D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins / Q000032:analysis*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells / Q000032:analysis; D006801:Humans; D007163:Immunosorbent Techniques; D007526:Isoelectric Point; D009698:Nucleoproteins / Q000032:analysis* / Q000276:immunology; D012259:Ribonuclease, Pancreatic / Q000378:metabolism
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D000939:Epitopes; D009698:Nucleoproteins; D003850:Deoxyribonuclease I; D012259:Ribonuclease, Pancreatic