The sample is detected on a western blot. By using electrophoretic mobility shift assay (EMSA), the activation profile of transcription factors can be detected. A multiplex approach for activation profiling is a TF chip system where several different transcription factors can be detected in parallel. The most commonly used method for identifying transcription factor binding sites is chromatin immunoprecipitation (ChIP). This technique relies on chemical fixation of chromatin with formaldehyde, followed by co-precipitation of DNA and the transcription factor of interest using an antibody that specifically targets that protein. The DNA sequences can then be identified by microarray or high-throughput sequencing (ChIP-seq) to determine transcription factor binding sites. If no antibody is available for the protein of interest, DamID may be a convenient alternative. Classes As described in more detail below, transcription factors may be classified by their (1) mechanism of action, (2) regulatory function, or (3) sequence homology (and hence structural similarity) in their DNA-binding domains. Mechanistic There are two mechanistic classes of transcription factors: General transcription factors are involved in the formation of a preinitiation complex. The most common are abbreviated as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. They are ubiquitous and interact with the core promoter region surrounding the transcription start site(s) of all class II genes. Upstream transcription factors are proteins that bind somewhere upstream of the initiation site to stimulate or repress transcription. These are roughly synonymous with specific transcription factors, because they vary considerably depending on what recognition sequences are present in the proximity of the gene. Functional Transcription factors have been classified according to their regulatory function: I. constitutively active – present in all cells at all times – general transcription factors, Sp1, NF1, CCAAT II. conditionally active – requires activation II.A developmental (cell specific) – expression is tightly controlled, but, once expressed, require no additional activation – GATA, HNF, PIT-1, MyoD, Myf5, Hox, Winged Helix II.B signal-dependent – requires external signal for activation II.B.1 extracellular ligand (endocrine or paracrine)-dependent – nuclear receptors II.B.2 intracellular ligand (autocrine)-dependent – activated by small intracellular molecules – SREBP, p53, orphan nuclear receptors II.B.3 cell membrane receptor-dependent – second messenger signaling cascades resulting in the phosphorylation of the transcription factor II.B.3.a resident nuclear factors – reside in the nucleus regardless of activation state – CREB, AP-1, Mef2 II.B.3.b latent cytoplasmic factors – inactive form reside in the cytoplasm, but, when activated, are translocated into the nucleus