Purification of sequence-specific DNA-binding proteins by affinity chromatography

• Published in: Current protocols in molecular biology (Print) Vol. Chapter 12; p. Unit 12.10
• Main Authors: Kerrigan, L A, Kadonaga, J T
• Format: Journal Article
• Language: English
• Published: United States 01.05.2001
• Subjects: Animals; Binding, Competitive; Chromatography, Affinity; Chromatography, Agarose; Cyanogen Bromide; DNA - chemistry; DNA - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; Electrophoresis, Agar Gel; Electrophoresis, Polyacrylamide Gel; Humans; Protein Binding; Sepharose - analogs & derivatives
• ISSN: 1934-3647
• DOI: 10.1002/0471142727.mb1210s24

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 basic protocol describes preparation of a DNA affinity resin, including cyanogen bromide (CNBr) activation of the agarose support. An 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 basic protocol outlines the affinity chromatography procedure. A second support protocol 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.