Chapter 13: Analysis of Protein-DNA Interactions

This analysis is done after cis elements of promoter have been suggested but still don’t know what they are, few fragments with potential cis elements but consider the notion that many transcriptional factors bind as dimers

Electrophoretic Mobility Shift Assay

  • Label DNA fragment with potential cis element labelled radioactively because ethidium bromide will not detect
  • Mix with protein with potential trans factor
  • Electrophorese the fragment alone, product of DNA + protein, and DNA + protein + antibody

DNA probe binding to the protein will be bigger and higher on the gel, antibody and protein will be highest. To identify whether the protein is binding specifically to the probe, include a mutant probe and see if the shift occurs. There will always be a probe line as it is in excess

Control: Add non-competitive competitor before addition of probe so proteins bind it, add more WT unlabelled probe which should decrease the interaction, then add unlabelled mutant probe which shouldn’t change interaction

Suitable for any DNA protein interactions, can detect multiple proteins to one fragment, test essential nt for binding with mutants, but cannot record the complex from the gel

DNase 1 Footprinting

Will reveal the region of DNA bound by a purified protein and determine exact nt

  • Isolate fragment containing cis element (PCR) and label it radioactively on one end (T4 polynucleotide kinase but since only on one end, then add RE at another end and cut it off)
  • Mix with extract containing potential DNA binding protein and add dilute DNase1 so each DNA fragment is only cut once, should give a ladder of fragments differing by one nt
  • Electrophorese on high resolution gel and compare migration with fragment not mixed with protein, some regions od DNA are protected by the binding or protein so were not cut
  • Control: Leave protein out of reaction

Suitable to test any protein DNA interaction and can detect multiple proteins to one fragment, test essential nt for binding with mutants but not sensitive since depends on ability of DNase 1 to access DNA and many TF bind to many sites so need the purified protein of interest for the assay

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