Protocols • Discovery of Prospective Regulatory Transcription Factors

[Under Construction] This set of protocols covers the Scott group work on discovery of regulatory TFs. Pull-down, identification, recombinant expression, binding site characterization, and functional validation are covered.

Differential DNA Pull-down of Prospective TFs [.ppt revised 2005.06.21 by M.Wang]

• Magnetic bead-DNA preparation, handling
• Cell extract incubation, washing, elution
• PAGE separation

MS Protein Identification of Prospective TFs

• In-gel trypsin digestion
  • Destaining (silver / coommassie / SYPRO ruby)
  • Reduction & alkylation
• Peptide mass mapping

Clone, Express, Purify Prospective TFs

• pET Vector Cloning (see Primer on Cloning, Expression, Purification)
• Gateway Cloning

Biophysical Identification of TF Binding Site

• Electromobility shift assay (EMSA) [.pdf revised 2006.08.21 by G.Lipscomb]
• DNA fooprinting
  • DNase I
  • Hydroxyl radical
• Systematic evolution of ligands by exponential enrichment (SELEX)

Regulated in vitro Transcriptional Profile

• Reconstitution of recombinant Pf RNA polymerase
• Non-specific transcription assay
• Template/promoter-specific transcription assay
• Regulated transcription assay

scott@chem.uga.edu
Department of Chemistry
University of Georgia
Athens, GA 30602-2556
706 542-2240 | FAX: 706 542-2295