UNIT 12.10 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 Molecular Biology
How to Cite
Kerrigan, L. A. and Kadonaga, J. T. 2001. Purification of Sequence-Specific DNA-Binding Proteins by Affinity Chromatography. Current Protocols in Molecular Biology. 24:12.10:12.10.1–12.10.18.
- Published Online: 1 MAY 2001
- Published Print: OCT 1993
Affinity chromatography is a very effective and straightforward means of purifying a protein based on its sequence-specific DNA-binding properties. 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. The first describes preparation of a DNA affinity resin, including cyanogen bromide (CNBr) activation of the agarose support. An Alternate Protocol provides a method to couple DNA to commercially available CNBr-activated Sepharose, and a support protocol describes how to purify crude synthetic oligonucleotides by gel electrophoresis prior to preparation of the affinity resin. The second outlines the affinity chromatography procedure. A second describes determination of 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.