Open AccessFluorescence-Based Protein Footprinting Using Histidine-Tagged Protein
Author(s) -
G.V. Rajendrakumar,
Sankar Adhya
Publication year - 1998
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/98241st04
Subject(s) - histidine , footprinting , biochemistry , protease , chemistry , dna footprinting , ligand (biochemistry) , binding site , dna , microbiology and biotechnology , biology , dna binding protein , enzyme , transcription factor , gene , receptor , base sequence
We describe a procedure for protein footprinting to identify the region(s) of a protein that interacts with a ligand. The method utilized the affinity of a stretch of histidine residues cloned into the protein to metalchelated resin. After limited protease digestion, the histidine-tagged end fragments were separated by the resin and labeled with a fluorescein derivative. Resolving the labeled digestion products on a denaturing polyacrylamide gel and visualizing the peptides using a FluorImager™ provided a way to identify the protease target sites that were protected from digestion because of interaction with DNA. The protection experiments would be applicable not only to detect direct contact sites but also sites allosterically altered by ligand binding.