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Journal of Histochemistry and Cytochemistry, Vol. 46, 1279-1290, November 1998, Copyright © 1998, The Histochemical Society, Inc.

ARTICLE

In Situ Staining for Poly(ADP–Ribose) Polymerase Activity Using an NAD Analogue

R. Eric Davisa, Veena Mysorea, Jared C. Browninga, Joseph C. Hsieha, Quynh-Anh T. Lub, and Peter D. Katsikisb
a Departments of Pathology, Stanford University Medical Center, Palo Alto, California
b Genetics, Stanford University Medical Center, Palo Alto, California

Correspondence to: R. Eric Davis, Metabolism Branch, Bldg 10, Rm 5A02, NIH, 10 Center Drive, MSC 1374, Bethesda, MD 20892-1374..

Poly(ADP-ribose) polymerase (PARP) is a highly abundant nuclear enzyme which metabolizes NAD, in response to DNA strand breakage, to produce chains of poly(ADP-ribose) attached to nuclear proteins. PARP activation has been implicated in ischemia/reperfusion injury, but its biological significance is not fully understood. We have modified an existing in situ method for detection of PARP activity by using an NAD analogue in which adenine is modified by an "etheno" (vinyl) bridge. Etheno–NAD serves as a PARP substrate in an initial enzymatic reaction; a specific antibody to ethenoadenosine is then used in an immunohistochemical reaction to detect the production of modified poly(ADP-ribose). The method produces strong and specific labeling of nuclei in which PARP has been activated, i.e., those in which DNA strand breaks have been produced, and the results can be analyzed by microscopy, flow cytometry, or colorimetry. The method is applicable to cultured cells in several formats and to frozen tissue sections. The particular characteristics of the new method may assist in future in situ studies of PARP activation. (J Histochem Cytochem 46:1279–1289, 1998)

Key Words: poly(ADP-ribose) polymerase, ADP-ribosyl transferase, EC 2.4.2.30, ethenoadenine, ethenoadenosine, 1,N6-etheno–NAD


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