PMID: 2492584Jan 6, 1989Paper

A dot-blot method for screening polyclonal and monoclonal antisera to poly(ADP-ribose)

Journal of Immunological Methods
S PrasadA Dritschilo

Abstract

ADP-ribosylation reactions play a key role at several points in cellular regulation and repair of DNA damage. The use of polyclonal or monoclonal antisera to poly(ADP-ribose) as probes to localize the site(s) of action of the polymer offers a promising tool for these studies. We report here a simple, sensitive method for detection and titration of these antisera to poly(ADP-ribose) using nitrocellulose membrane (NC) as a support for a dot-blot analysis. We take advantage of the fact that a highly labeled poly(ADP-ribose) preparation can be obtained by incubation of a 0.3 M KCl extract prepared from calf thymus nuclei with 32P-NAD. Such a preparation of labeled antigen is used as a reagent to detect the positive antibody spots on the NC with negligible background. Subsequent titration of the antisera and their semi-quantitative evaluation are also feasible using the dot-blot method. The sensitivity of the assay is only limited by the specific activity that can be achieved for the labeled polymer prepared as the antigen probe. The advantage of this method is that it eliminates the need to prepare pure, highly radiolabeled polymer as well as the fact that several samples can be handled on the membrane simultaneously. We demonstrat...Continue Reading

References

Apr 28, 1978·Biochemical and Biophysical Research Communications·P AdamietzH Hilz
Feb 1, 1985·The Journal of Clinical Investigation·R S Schwartz, B D Stollar
Sep 2, 1985·FEBS Letters·U Wintersberger, E Wintersberger
Nov 1, 1985·Developmental Biology·B W CherneyA I Caplan
Jul 10, 1974·Biochemical and Biophysical Research Communications·Y KanaiT Sugimura
May 1, 1983·Proceedings of the National Academy of Sciences of the United States of America·N MalikM Smulson

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Citations

Aug 2, 2016·Proceedings of the Japan Academy. Series B, Physical and Biological Sciences·Yoshiyuki Kanai
Jan 1, 1990·Autoimmunity·C NegriG C Astaldi Ricotti

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