Fluorescence-Based Protein Footprinting Using Histidine-Tagged Protein. (January 1998)

• Record Type: Journal Article
• Authors: Rajendrakumar, G.V.; Adhya, Sankar
• Is Part Of: Biotechniques. Volume 24:Number 1(1998)
• Page Start: 110
• Page End: 115
• Publication Date: 1998-01
• Subjects: Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/98241st04
• Languages: English
• ISSNs: 0736-6205

Abstract

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.