Transcription factor proteomics: identification by a novel gel mobility shift-three-dimensional electrophoresis method coupled with southwestern blot and high-performance liquid chromatography-electrospray-mass spectrometry analysis.

ABSTRACT: Transcription factor (TF) purification and identification is an important step in elucidating gene regulatory mechanisms. In this study, we present two new electrophoretic mobility shift assay (EMSA)-based multi-dimensional electrophoresis approaches to isolate and characterize TFs, using detection with either southwestern or western blotting and HPLC-nanoESI-MS/MS analysis for identification. These new techniques involve several major steps. First, EMSA is performed with agents that diminish non-specific DNA-binding and the DNA-protein complex is separated by native PAGE gel. The gel is then electrotransferred to PVDF membrane and visualized by autoradiography. Next, the DNA-protein complex, which has been transferred onto the blot, is extracted using a detergent-containing elution buffer

SUBMITTER: Jiang D 

PROVIDER: S-EPMC3174475 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature