Assay for protein modification by poly-ADP-ribose in vitro

Opeyemi A. Olabisi, Chi Wing Chow

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

The enzymatic function of poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP) is central to many of its function as a component of DNA repair machinery, modulator of gene transcription, and cell differentiation. While the auto-modification domain of PARP has been shown to be a primary acceptor site of poly-ADP ribose (pADPr), other DNA binding nuclear proteins are also modified by pADPr. It is -generally accepted that pADPr polymer is built upon the carboxyl side chain of specific Glu, Asp, and/or Lys residues within the target protein. Identification of the unique amino acid acceptors of pADPr in these nuclear proteins is an active area of study. Because of the heterogeneity of pADPr chain on modified -protein targets, the resulting modified proteins have unpredictable final masses, making it difficult to -identify acceptor amino acids. Using recombinant proteins, in vitro pADP ribosylation assay and mass spectrometry, we have been able to identify conserved Glu residue in transcription factor NFAT that is enzymatically modified in vitro with pADPr by PARP-1. We discuss this protocol here as a model approach for identifying pADPr acceptors in other nuclear proteins.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
Pages47-55
Number of pages9
Volume780
DOIs
StatePublished - 2011

Publication series

NameMethods in Molecular Biology
Volume780
ISSN (Print)10643745

Fingerprint

Poly Adenosine Diphosphate Ribose
Proteins
Nuclear Proteins
Amino Acids
Poly(ADP-ribose) Polymerases
In Vitro Techniques
DNA-Binding Proteins
Recombinant Proteins
DNA Repair
Cell Differentiation
Mass Spectrometry
Polymers
Transcription Factors

Keywords

  • Poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP)
  • Poly-ADP ribose (pADPr)
  • Transcription factor NFAT

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Olabisi, O. A., & Chow, C. W. (2011). Assay for protein modification by poly-ADP-ribose in vitro. In Methods in Molecular Biology (Vol. 780, pp. 47-55). (Methods in Molecular Biology; Vol. 780). https://doi.org/10.1007/978-1-61779-270-0_3

Assay for protein modification by poly-ADP-ribose in vitro. / Olabisi, Opeyemi A.; Chow, Chi Wing.

Methods in Molecular Biology. Vol. 780 2011. p. 47-55 (Methods in Molecular Biology; Vol. 780).

Research output: Chapter in Book/Report/Conference proceedingChapter

Olabisi, OA & Chow, CW 2011, Assay for protein modification by poly-ADP-ribose in vitro. in Methods in Molecular Biology. vol. 780, Methods in Molecular Biology, vol. 780, pp. 47-55. https://doi.org/10.1007/978-1-61779-270-0_3
Olabisi OA, Chow CW. Assay for protein modification by poly-ADP-ribose in vitro. In Methods in Molecular Biology. Vol. 780. 2011. p. 47-55. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-61779-270-0_3
Olabisi, Opeyemi A. ; Chow, Chi Wing. / Assay for protein modification by poly-ADP-ribose in vitro. Methods in Molecular Biology. Vol. 780 2011. pp. 47-55 (Methods in Molecular Biology).
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