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.
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.
Nozaki
T
T
Biochemistry Division, National Cancer Center Research Institute, Tokyo, Japan.
Masutani
M
M
Akagawa
T
T
Sugimura
T
T
Esumi
H
H
D001483:Base Sequence; D015153:Blotting, Western / Q000379:methods; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D011064:Poly Adenosine Diphosphate Ribose / Q000032:analysis / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011485:Protein Binding; D012313:RNA / Q000378:metabolism
D006657:Histones; D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D011064:Poly Adenosine Diphosphate Ribose; D012313:RNA; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases