An electrophoretic mobility shift assay (EMSA) is a common and invaluable technique which can be utilized to study the affinity of proteins to a specific DNA or RNA sequence. These assays are performed in vitro with protein extracts isolated from either cultured cells or isolated tissues. Here, we describe the methodology used to isolate the cytoplasmic and nuclear protein extracts from both cultured cells and tissues and utilize the nuclear protein fraction to assess NF-κB DNA-binding activity by EMSA analysis.