Non-covalent interaction between poly(ADP-ribose) and cellular proteins: an application of a poly(ADP-ribose)-western blotting method to detect poly(ADP-ribose) binding on protein-blotted filter

Biochem Biophys Res Commun. 1994 Jan 14;198(1):45-51. doi: 10.1006/bbrc.1994.1007.

Abstract

We describe a sensitive method for the detection of interactions between poly(ADP-ribose) and proteins. Proteins were blotted onto nitrocellulose filters and incubated with 32P-labeled poly(ADP-ribose). Purified core histones and poly(ADP-ribose) polymerase were found to bind poly(ADP-ribose) polymer. Blots of HeLa cell protein extracts revealed a 48 kDa protein and several others of smaller than 35 kDa likewise bound the polymers even at high salt concentrations. Those proteins, along with a 69 kDa protein, also showed resistance to competitor DNA. Polymer binding of aforesaid HeLa extract proteins was restricted to polymers above 20 residues in length. Thus poly(ADP-ribose)-protein affinities were polymer-length dependent.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Blotting, Western / methods
  • DNA / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • HeLa Cells
  • Histones / metabolism*
  • Humans
  • Molecular Sequence Data
  • Molecular Weight
  • Oligodeoxyribonucleotides
  • Phosphorus Radioisotopes
  • Poly Adenosine Diphosphate Ribose / analysis
  • Poly Adenosine Diphosphate Ribose / metabolism*
  • Poly(ADP-ribose) Polymerases / metabolism*
  • Protein Binding
  • RNA / metabolism

Substances

  • Histones
  • Oligodeoxyribonucleotides
  • Phosphorus Radioisotopes
  • Poly Adenosine Diphosphate Ribose
  • RNA
  • DNA
  • Poly(ADP-ribose) Polymerases