Single molecule Förster resonance energy transfer (smFRET) is a powerful biophysical structural method for investigating the structural dynamics and interactions of biomolecules at the single-molecule level. By utilizing fluorophores strategically placed on proteins, smFRET enables the measurement of distance changes between specific sites, providing insights into conformational changes, binding events, and molecular interactions. In the Komives lab, smFRET has been utilized to prove the dynamics of Nuclear Factor-kappa B (NFκB), a pivotal transcription factor involved in immune response and inflammation. Specifically, studies have focused on the NFκB p50/RelA heterodimer, revealing valuable insights into its conformational changes and DNA binding dynamics. The Komives lab was able to purify fulllength NFκB, including the intrinsically disordered transcription activation domain (TAD), and found that the TAD enhances the binding affinity of NFκB to DNA, with effect being more pronounced in binding to nonconsensus DNA sequences. However, a significant knowledge gap remains regarding the binding and dissociation mechanism of NFκB during DNA binding in the presence of multiple nearby binding sites. For my thesis I propose addressing this gap by combining smFRET techniques with optical tweezers using the Lumicks C-Trap system to visualize both the DNA substrate (lambda DNA) and the NFκB. This integrative approach holds promise for advancing our understanding of NFκB mediated transcriptional regulation and may offer new avenues for therapeutic intervention in diseases associated with dysregulated NFκB signaling pathways. I propose to do this by first establishing a working prism-based smFRET TIRF microscope, which hadn’t been working for two years prior to my work with it. Then using the established set up to investigate interdomain motions of fulllength NFκB, and combining this technique with Lumicks® C-Trap system to investigate the binding mechanism of NFκB.