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Updated: Jul 19, 2025
Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes
Published on: September 28, 2018
Using Affinity Pulldown Assays to Study Protein-Protein Interactions of Human NEIL1 Glycosylase and the Checkpoint
Drew T McDonald1,2, Pam S Wang1,3, Jennifer Moitoza Johnson1,4
1Department of BioEngineering and BioMedical Sciences, Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
Abstract:
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.
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