Kits that contain a biotinylated DNA oligonucleotide, a capture antibody, and Streptavidin HRP to demonstrate relative transcription factor activity.
A biotinylated double stranded (ds) oligonucleotide containing a specific consensus transcription factor binding site is incubated with nuclear extracts.
Transcription factor molecules are bound by ds oligonucleotides.
Transcription factor-ds oligonucleotide complexes are subsequently captured by an immobilized antibody specific for the active transcription factor.
After washing away unbound material, detection utilizing Streptavidin-HRP is performed. An unlabeled ds competitor oligonucleotide is provided to demonstrate the specificity of the assay.
Tetramethylbenzidine (TMB) substrate solution is added to the wells and a blue color develops in proportion to the amount of analyte present in the sample. Color development is stopped, turning the color in the wells to yellow.
Note: This assay may be converted to measure total levels of a specific transcription factor levels by substituting the appropriate antibody.