UNIT 9.6 Purification of Sequence-Specific DNA-Binding Proteins by Affinity Chromatography
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Protein Science
How to Cite
Kerrigan, L. A. and Kadonaga, J. T. 2001. Purification of Sequence-Specific DNA-Binding Proteins by Affinity Chromatography. Current Protocols in Protein Science. 9.6.1–9.6.18.
- Published Online: 1 MAY 2001
- Published Print: MAR 1998
The affinity chromatography procedure described in this unit uses DNA containing specific recognition sites for the desired protein that has been covalently linked to a solid support. Preparation of a DNA affinity resin, including cyanogen bromide (CNBr) activation of the agarose support, is described, and an alternate protocol provides a method to couple DNA to commercially available CNBr-activated Sepharose. A method for purification of crude synthetic oligonucleotides by gel electrophoresis prior to preparation of the affinity resin is also provided. A detailed protocol for the actual affinity chromatography procedure is described and a support protocol allows the investigator to determine the appropriate type and quantity of nonspecific competitor DNA that should be used in the procedure and its preparation. Parameters essential to the success of an affinity chromatography experiment are discussed in detail in the Commentary.