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Purification and N-terminal amino acid sequence of proliferating cell nuclear antigen (PCNA)/cyclin and development of ELISA for anti-PCNA antibodies

Academic Article
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Overview

authors

  • Ogata, K.
  • Ogata, Y.
  • Nakamura, R. M.
  • Tan, Eng

publication date

  • 1985

journal

  • Journal of Immunology  Journal

abstract

  • Proliferating cell nuclear antigen (PCNA), also called cyclin, was purified from PBS extract of rabbit thymus by using a combination of ammonium sulfate fractionation, DEAE-Sephacel, HPLC ion exchange, and HPLC gel filtration column chromatography. PCNA was purified more than 600 times and was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. SDS-PAGE showed that a 36 kD protein was selectively isolated in this purification process, and this protein was identified as PCNA by immunoblotting. Other previously identified nuclear antigens, Sm, nRNP, SS-A/Ro, SS-B/La, histone, and DNA, were not detected in this preparation by counterimmunoelectrophoresis and enzyme-linked immunosorbent assay (ELISA). Purified PCNA was used as an antigen to develop ELISA for rapid and specific detection of anti-PCNA in human sera. For further purification, the 36 kD band was electrophoretically eluted from SDS gel slices. The amino acid composition and the first 25 residues from the N-terminus of the protein were determined by using electroeluted PCNA. This amino acid sequence was found to be unique and showed little sequence homology with existent proteins in the protein identification resources databank.

subject areas

  • Amino Acid Sequence
  • Amino Acids
  • Antigen-Antibody Reactions
  • Autoantibodies
  • Chromatography, Gel
  • Chromatography, High Pressure Liquid
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • Immunoenzyme Techniques
  • Lupus Erythematosus, Systemic
  • Molecular Weight
  • Nucleoproteins
  • Proliferating Cell Nuclear Antigen
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Identity

International Standard Serial Number (ISSN)

  • 0022-1767

PubMed ID

  • 2863307
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Additional Document Info

start page

  • 2623

end page

  • 2627

volume

  • 135

issue

  • 4

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