Reporter assay using the beta-galactosidase (lacZ) gene. The lacZ gene is typically fused to the promoter of interest. Differential regulation of the promoter mediated by the TF is assessed by induction of the system and evaluation of lacZ expression. Bacteria expressing lacZ appear blue when grown on a X-gal medium. The assay is often performed using a plasmid borne construction on a lacZ(def) strain.

MSA can be used to align promoter regions and highlight regions of conservation that may be indicative of transcription factor binding sites.

Non-targeted mutagenesis. Typically accomplished in vivo by means of radiation or a DNA-damaging agent, or in vitro using degenerate PCR. Not frequently used in TF-binding site determination. It is sometimes used to investigate the effect of mutations in transcription factor binding domains, in conjunction with expression assays.

Visual inspection of promoter region to identify putative binding sites based on previous knowledge