TY - CHAP
T1 - Fluorescence-Based Analyses of Poly(ADP-Ribose) Length by Gel Electrophoresis, High-Performance Liquid Chromatography, and Capillary Electrophoresis
AU - Badiee, Mohsen
AU - Boutonnet, Audrey
AU - Phan, Dat
AU - Leung, Anthony K.L.
N1 - Funding Information:
We acknowledge the funding support for this work from the National Institutes of Health grants T32-CA009110 (M.B.), R01-GM104135 (AKLL), and RF1-AG071326 (AKLL).
Publisher Copyright:
© 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2023
Y1 - 2023
N2 - 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.
AB - 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.
KW - Capillary electrophoresis
KW - Fluorescent detection
KW - High-performance liquid chromatography
KW - Poly(ADP-ribose)
KW - Poly(ADP-ribose) chain length analysis
KW - Polyacrylamide gel electrophoresis
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U2 - 10.1007/978-1-0716-2891-1_1
DO - 10.1007/978-1-0716-2891-1_1
M3 - Chapter
C2 - 36515826
AN - SCOPUS:85144587108
T3 - Methods in Molecular Biology
SP - 3
EP - 21
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -