Purification and characterization of transcription factors.

Transcription factors (TFs) are essential for the expression of all proteins, including those involved in human health and disease. However, TFs are resistant to proteomic characterization because they are frequently masked by more abundant proteins due to the limited dynamic range of capillary liquid chromatography-tandem mass spectrometry and protein database searching. Purification methods, particularly strategies that exploit the high affinity of TFs for DNA response elements (REs) on gene promoters, can enrich TFs prior to proteomic analysis to improve dynamic range and penetrance of the TF proteome. For example, trapping of TF complexes specific for particular REs has been achieved by recovering the element DNA-protein complex on solid supports. Additional methods for improving dynamic range include two- and three-dimensional gel electrophoresis incorporating electrophoretic mobility shift assays and Southwestern blotting for detection. Here we review methods for TF purification and characterization. We fully expect that future investigations will apply these and other methods to illuminate this important but challenging proteome.