When proteins are properly purified, experimental results hold the distinct advantage of directly observing protein behavior without concern that unknown factors influence the results. Furthermore, protein purification has previously been an obligate requirement for numerous types of biophysical analyses, ranging from enzyme kinetics and structural studies to experiments where protein behavior is monitored at the single-molecule level [1], [2], [3], [4]. With the advent of new single-molecule techniques [5], [6], [7], [8], [9], [10], [11], [12], such as the Single-molecule Analysis of DNA-binding proteins from Nuclear Extracts (SMADNE), the necessity to always purify proteins has been lifted in order to study proteins at the single-molecule level [5]. Utilizing nuclear extracts directly expressed from mammalian cells has a number of advantages such as: rapidly screening variants, post-translational modifications (PTMs) can be preserved, proteins that are traditionally challenging [13], [14] to express/purify from bacteria can be readily obtained within minutes of lysing cells, purification of large mammalian complexes is readily amenable from extracts, and using lysates does not require the hours if not days of time necessary to fully purify protein. At the same time, experiments like SMADNE that use nuclear extracts hold the dual advantage and disadvantage that many of the thousands of proteins present in a nucleus are also present in the experiment, albeit at ∼500-fold lower concentrations than in the nucleus, which may make the results difficult to interpret. Though, the presence of other proteins is also beneficial because the experimental results may be more indicative of behavior in a biological context compared to a protein studied in isolation. However, this reward holds the caveat that the identities of these unseen “dark” proteins are unknown until follow-up experiments are performed, such as fluorescent labeling or knocking down putative interacting partners [5].

8-oxoguanine glycosylase 1 (OGG1) is a key protein involved in the repair of the oxidative damage 8-oxoguanine (8-oxoG) during base excision repair (BER), where OGG1 identifies 8-oxoG across from a cytidine and subsequently cleaves the glycosidic bond to leave behind an abasic site [15]. Similar to all DNA glycosylases, OGG1 faces the extraordinary challenge of rapidly identifying 8-oxoG amongst billions of undamaged DNA base pairs [16]. Thus, it has been proposed and observed that OGG1 diffuses along the DNA helix to aid in its search for damage [17], [18]. The most direct way to understand the damage search process of OGG1 is fluorescent labeling of the protein and observing its movement on DNA in real time. Importantly, OGG1 (and bacterial DNA-formamidopyrimidine glycosylase, FPG) have been extensively characterized at the single-molecule level using multiple different imaging techniques [5], [17], [19]. Additionally, OGG1 has also been characterized on a variety of nucleic acid substrates including undamaged DNA [17], DNA containing abasic sites [18], and DNA containing oxidative damage [5]. Finally, OGG1 has been labeled with numerous fluorescent labeling strategies, including Cy3 maleimide labeling, Qdot conjugation with an antibody, and fusing a fluorescent fusion protein to the protein of interest, making it a great model system for single-molecule studies [5], [17], [18].

The purpose of this article is two-fold, to review the SMADNE approach and to compare the behavior of a purified protein versus the SMADNE approach using OGG1. In our experiments, we utilized OGG1-GFP as a model system to determine how nuclear proteins present in extracts may alter single-molecule binding kinetics. Three different contexts were assayed: (1) OGG1-GFP purified from a bacterial expression system, (2) purified OGG1-GFP spiked into the nuclear extracts with no overexpressed protein, and (3) OGG1-GFP overexpressed in nuclear extracts without purification (SMADNE approach). This has allowed us to directly compare and contrast these three different experimental systems, while also beginning to deconvolute the role of unknown nuclear proteins in the DNA damage search and detection process.