Protein-DNA and protein-protein interactions play essential roles in many biologic processes such as transcription, replication, recombination, and DNA repair. One of the most popular approaches to investigate specific protein-nucleic acid interactions is the electrophoretic mobility shift assay (EMSA). We have developed a new nonradioactive method to detect both nucleotides and protein in EMSA. Nucleic acids are detected with SYBR(R) Green EMSA dye, while proteins are subsequently detected with SYPRO(R) Ruby EMSA dye. All fluorescent staining steps are performed after the entire gel-shift experiment is completed, so there is no need to prelabel either the nucleic acids or the protein. The two-color fluorescence dye staining procedure is fast, simple, and allows independent quantitative determination of either free or bound nucleic acids and protein. The interactions between lac repressor-operator, and among the T4 primase-helicase, primase-DNA, helicase-DNA, and within T4 [ssDNA-primase-helicase(6)] primosome, were used to demonstrate the advantages of this two-color fluorescence detection EMSA method.