Purification and characterization of transcription factors

Abstract

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 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 response elements 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.

Proteomic Characterization of Transcription and Splicing Factors Associated with a Metastatic Phenotype in Colorectal Cancer

“…However, TFs are resistant to proteomic characterization because they are frequently masked by more abundant proteins . Less than 5% of the TFs have ever been purified and characterized . The very low abundance of TFs complicates their purification, but new techniques are becoming available to make purification and analysis much less challenging and more rational .…”

“…3 Less than 5% of the TFs have ever been purified and characterized. 4 The very low abundance of TFs complicates their purification, but new techniques are becoming available to make purification and analysis much less challenging and more rational. 5 The high affinity of TFs for REs on gene promoters enabled the enrichment for low abundance TFs prior to proteomic analysis.…”

Differential expression of CYB5A in Chinese and European pig breeds due to genetic variations in the promoter region

“…), which are essential for protein expression (Nagore et al . ). The pGL3‐hapB construct showed an approximately sixfold increase in reporter activity, similar to the qPCR results from liver samples.…”

Syntaxin 1A Gene Is Negatively Regulated in a Cell/Tissue Specific Manner by YY1 Transcription Factor, Which Binds to the −183 to −137 Promoter Region Together with Gene Silencing Factors Including Histone Deacetylase

“…The isolation of DNA–protein complexes based on streptavidin and biotin interaction was adapted from reference [ 14 , 15 ] with modifications. Magnosphere MS300 (TaKaRa, Shiga, Japan) streptavidin magnetic beads (1 mg) were incubated for 30 min at 25 °C with 1 nmol biotinylated OL2 DNA in 0.4 ml of binding buffer (5 mM Tris-HCl, pH 7.5, 1 M NaCl, 0.5 mM EDTA and 0.05% Tween20).…”