Utilizing nuclear extracts to characterize protein: DNA interactions at the single molecule level.

Single molecule experiments are invaluable approaches to analyze the dynamics of protein-protein and protein-DNA interactions in real time. SMADNE, single molecule analysis of DNA binding proteins from nuclear extracts, is a new method that allows analysis of a fluorescently tagged overexpressed protein of interest near its native environment while still retaining the advantages of single molecule approaches. Having all the endogenous proteins found in the nucleus provides more biologically relevant information due to their interactions with the protein of interest. Examples of such include the ability for posttranslational modifications to occur, intrinsic stabilization factors, and high labeling efficacy of the protein of interest. Furthermore, having the capabilities to incorporate different DNA substrates and protein variants can elucidate information of the system in a more detailed manner. Finally, orthogonal labeling strategies allows determination of the order of assembly and disassembly of several proteins at sites of damage. This chapter will describe the methodologies, benefits, and applications of SMADNE.