Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates.

ABSTRACT: Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5' hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficul

SUBMITTER: Pillon MC 

PROVIDER: S-EPMC7389563 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature