Comparison of CRISPR Genomic Tagging for Affinity Purification and Endogenous Immunoprecipitation Coupled with Quantitative Mass Spectrometry To Identify the Dynamic AMPKα2 Interactome.

ABSTRACT: Recent advances in genome editing technologies have enabled the insertion of epitope tags at endogenous loci with relative efficiency. We describe an approach for investigation of protein interaction dynamics of the AMP-activated kinase complex AMPK using a catalytic subunit AMPKα2 (PRKAA2 gene) as the bait, based on CRISPR/Cas9-mediated genome editing coupled to stable isotope labeling in cell culture, multidimensional protein identification technology, and computational and statistical analyses. Furthermore, we directly compare this genetic epitope tagging approach to endogenous immunoprecipitations of the same gene under homologous conditions to assess differences in observed interactors. Additionally, we directly compared each enrichment strategy in the genetically modified cell

SUBMITTER: Stein BD 

PROVIDER: S-EPMC7512655 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature