Fluorescence-Based Analyses of Poly(ADP-Ribose) Length by Gel Electrophoresis, High-Performance Liquid Chromatography, and Capillary Electrophoresis.

Methods Mol Biol

Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.

Published: December 2022

AI Article Synopsis

  • Poly(ADP-ribose) (PAR) is a polymer consisting of ADP-ribose units and is crucial for processes like DNA repair and RNA metabolism.
  • Recent findings show that the length of PAR is carefully regulated and affects how it interacts with proteins.
  • The study introduces fluorescent methods, particularly a new capillary electrophoresis technique, to effectively measure and analyze PAR length, offering a faster and more automated alternative to traditional methods.

Article Abstract

Poly(ADP-ribose) (PAR) is a homopolymer made of two or more adenosine diphosphate ribose (ADP-ribose) units. The polymer is usually conjugated to protein as a posttranslational modification playing key roles in cellular processes, such as DNA repair, RNA metabolism, and biomolecular condensate formation. Emergent data revealed that PAR length is highly regulated and determines the selection of and affinity towards protein binders. Here, we describe several fluorescence-based methods that quantify PAR length distributions. Briefly, we use the bioconjugation technique ELTA (enzymatic labeling of terminal ADP-ribose) to fluorescently label PAR, which can be isolated from in vitro and cellular samples. We describe a novel capillary electrophoresis method to separate and quantify PAR length and compare the profile to gel electrophoresis- and high-performance liquid chromatography-based methods. The capillary electrophoresis method is rapid and automatable, enabling accurate determination of the length profiles from subfemtomole quantities of PAR.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10281322PMC
http://dx.doi.org/10.1007/978-1-0716-2891-1_1DOI Listing

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