CREB (cAMP-response-element-binding protein) is a transcription factor that binds to cAMP-responsive element (CRE) promoter sites to regulate the transcription of numerous genes involved in metabolic regulation, depression, long term memory, and other physiological processes. Phosphorylation on serine 133 (Ser133) activates CREB to induce transcription of target genes. Diverse stimuli such as growth factors, neurotransmitters, hypoxia, growth factors, UV light, survival signals, and stress signals are some of the known activators of CREB. The signaling systems and mechanisms involved in CREB activation continue to be of interest in academic research and drug discovery programs. Cayman’s CREB (Phospho-Ser133) Transcription Factor Assay is a non-radioactive, sensitive method for detecting CREB DNA binding activity. CREB contained in a nuclear extract or whole cell lysate binds specifically to the DNA cAMP response element immobilized to the wells a 96-well plate. The activated CREB transcription factor complex is detected by addition of a specific primary antibody directed against Phospho-Ser133 on CREB. A secondary antibody conjugated to HRP is used to provide a sensitive colorimetric readout at 450 nm.