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Many proteins sequence-specifically bind duplex DNA, e.g., transcriptional regulatory proteins. Analysis of their interactions can be performed by a variety of methods, including electrophoretic mobility shift assays (EMSA) and quantitative DNase I footprinting. Here we describe an additional electrophoretic method, restriction endonuclease protection assays (REPA), to qualitatively and quantitatively study the interactions of thermophilic transcription regulatory proteins to PCR-generated, infrared-fluorescent DNA probes. REPA utilizes type IIS restriction endonucleases (IISRE), which cleave double-stranded DNA without specificity at a fixed distance from their recognition sequence. Thus, IISREs can be used to probe the occupancy of a suitably situated DNA-binding site for a variety of ligands. REPA has certain advantages as it does not require the maintenance of ligand-DNA complex stability during gel electrophoresis, as is the case with EMSA and is technically far less challenging than quantitative DNase I footprinting.