Unit

UNIT 9.7 Purification of DNA-Binding Proteins Using Biotin/Streptavidin Affinity Systems

  1. Lewis A. Chodosh1,
  2. Stephen Buratowski2

Published Online: 1 MAY 2001

DOI: 10.1002/0471140864.ps0907s12

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Chodosh, L. A. and Buratowski, S. 2001. Purification of DNA-Binding Proteins Using Biotin/Streptavidin Affinity Systems. Current Protocols in Protein Science. 12:9.7:9.7.1–9.7.13.

Author Information

  1. 1

    University of Pennsylvania, Philadelphia, Pennsylvania

  2. 2

    Harvard Medical School, Boston, Massachusetts

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: JUN 1998

Abstract

This unit presents purification protocols that exploit the tight and essentially irreversible complex that biotin forms with streptavidin. A DNA fragment containing a high-affinity binding site for the protein of interest is prepared and a molecule of biotinylated nucleotide is incorporated into one of the ends of the DNA fragment. The protein of interest is allowed to bind to the high-affinity recognition site present in the biotinylated fragment. The tetrameric protein streptavidin is then bound to the biotinylated end of the DNA fragment. Next, the protein/biotinylated fragment/streptavidin ternary complex is efficiently removed by adsorption onto a biotin-containing resin. Since streptavidin is multivalent, it is able to serve as a bridge between the biotinylated DNA fragment and the biotin-containing resin. Proteins remaining in the supernatant are washed away under conditions that maximize the stability of the DNA-protein complex. Finally, the protein of interest is eluted from the resin with a high-salt buffer. Both batch and column formats are presented, as is a protocol for the use of streptavidin-agarose. A support protocol describes a mobility shift assay for detecting sequence-specific DNA-binding proteins.