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  • Generation and Characterization of Recombinant Antibody-like ADP-Ribose Binding Proteins.

Generation and Characterization of Recombinant Antibody-like ADP-Ribose Binding Proteins.

Biochemistry (2017-10-21)
Bryan A Gibson, Lesley B Conrad, Dan Huang, W Lee Kraus
ABSTRACT

ADP-ribosylation is an enzyme-catalyzed post-translational modification of proteins in which the ADP-ribose (ADPR) moiety of NAD+ is transferred to a specific amino acid in a substrate protein. The biological functions of ADP-ribosylation are numerous and diverse, ranging from normal physiology to pathological conditions. Biochemical and cellular studies of the diverse forms and functions of ADPR require immunological reagents that can be used for detection and enrichment. The lack of a complete set of tools that recognize all forms of ADPR [i.e., mono-, oligo-, and poly(ADP-ribose)] has hampered progress. Herein, we describe the generation and characterization of a set of recombinant antibody-like ADP-ribose binding proteins, in which naturally occurring ADPR binding domains, including macrodomains and WWE domains, have been functionalized by fusion to the Fc region of rabbit immunoglobulin. These reagents, which collectively recognize all forms of ADPR with different specificities, are useful in a broad array of antibody-based assays, such as immunoblotting, immunofluorescent staining of cells, and immunoprecipitation. Observations from these assays suggest that the biology of ADPR is more diverse, rich, and complex than previously thought. The ARBD-Fc fusion proteins described herein will be useful tools for future exploration of the chemistry, biochemistry, and biology of ADP-ribose.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-pan-ADP-ribose binding reagent, from Escherichia coli
Sigma-Aldrich
Anti-poly-ADP-ribose binding reagent, Anti-poly-ADP-ribose binding reagent is a reagent that selectively binds to ADP ribose for use in Western Blotting, Immunocytochemistry and Dot Blot.
Sigma-Aldrich
Anti-mono- and poly-ADP-ribose binding reagent, from Escherichia coli
Sigma-Aldrich
Anti-mono- ADP-ribose binding reagent, from Escherichia coli