Immunological determination and size characterization of poly(ADP-ribose) synthesized in vitro and in vivo.
Poly(ADP-ribose) polymerase is a DNA break detecting enzyme playing a role in the surveillance of genome integrity. Poly(ADP-ribose) is synthesized rapidly and transiently from beta-NAD in response to DNA damaging agents. In order to study the physiological significance of poly(ADP-ribose) metabolism, we have developed immunological methods which enable us to study endogenous poly(ADP-ribose) without interfering with cell metabolism and integrity. For this purpose, we produced a highly specific polyclonal anti-poly(ADP-ribose) antibody which immunoreacts with polymers and oligomers. In addition to the immunodot blot method recently described by us (Affar et al., Anal. Biochem. 259 (1998) 280-283), other applications were investigated in cells: (i) detection of poly(ADP-ribose) by ELISA; (ii) characterization of poly(ADP-ribose) size using high resolution gel electrophoresis of polymers, followed by its transfer onto a positively charged membrane and detection with anti-poly(ADP-ribose) antibody; (iii) immunocytochemistry and flow cytometry analyses allowing poly(ADP-ribose) study at the level of individual cells.
Affar
E B
EB
Poly(ADP-ribose) Metabolism Group, Health and Environment Unit, Laval University Medical Research Center, CHUQ, 2705, Boul. Laurier, Ste-Foy, Quebec G1V 4G2, Canada.
Duriez
P J
PJ
Shah
R G
RG
Winstall
E
E
Germain
M
M
Boucher
C
C
Bourassa
S
S
Kirkland
J B
JB
Poirier
G G
GG
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D002460:Cell Line; D005591:Chemical Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D015151:Immunoblotting; D007150:Immunohistochemistry; D008769:Methylnitronitrosoguanidine; D051379:Mice; D011064:Poly Adenosine Diphosphate Ribose / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology
D000906:Antibodies; D008769:Methylnitronitrosoguanidine; D011064:Poly Adenosine Diphosphate Ribose