Using Affinity Pulldown Assays to Study Protein-Protein Interactions of Human NEIL1 Glycosylase and the Checkpoint Protein RAD9-RAD1-HUS1 (9-1-1) Complex.

Affinity pulldown is a powerful technique to discover novel interaction partners and verify a predicted physical association between two or more proteins. Pulldown assays capture a target protein fused with an affinity tag and analyze the complexed proteins. Here, we detail methods of pulldown assays for two high-affinity peptide fusion tags, Flag tag (DYKDDDDK) and hexahistidine tag (6xHis), to study protein-protein interactions of human NEIL1 glycosylase and the checkpoint protein complex RAD9-RAD1-HUS1 (9-1-1). We uncover unique interactions between 9-1-1 and NEIL1, which suggest a possible inhibitory role of the disordered, phosphorylated C-terminal region of RAD9 in regulating NEIL1 activity in base excision repair through lack of physical association of 9-1-1 and NEIL1. © 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Citation

Drew T McDonald, Pam S Wang, Jennifer Moitoza Johnson, Miaw-Sheue Tsai. Using Affinity Pulldown Assays to Study Protein-Protein Interactions of Human NEIL1 Glycosylase and the Checkpoint Protein RAD9-RAD1-HUS1 (9-1-1) Complex. Methods in molecular biology (Clifton, N.J.). 2023;2701:199-207

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PMID: 37574484

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