Unit

UNIT 12.7 Detection, Purification, and Characterization of cDNA Clones Encoding DNA-Binding Proteins

  1. Harinder Singh

Published Online: 1 MAY 2001

DOI: 10.1002/0471142727.mb1207s13

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Singh, H. 2001. Detection, Purification, and Characterization of cDNA Clones Encoding DNA-Binding Proteins. Current Protocols in Molecular Biology. 13:12.7:12.7.1–12.7.10.

Author Information

  1. University of Chicago, Chicago, Illinois

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: JAN 1991

Abstract

In this unit, an appropriate recombinant clone is detected in an expression library with a DNA recognition-site probe, purified, and shown to encode a DNA-binding domain of defined sequence specificity. The strategy described below obviates purification of a sequence-specific DNA-binding protein for the purpose of isolating its gene; it simply requires a suitable cDNA library constructed in the expression vector lgt11 and a DNA recognition-site probe. The basic protocol enables the detection and purification of clones encoding sequence-specific DNA-binding proteins. The alternate protocol describes a denaturation/renaturation procedure that can increase detection of certain clones. The support protocol provides a rapid means of characterizing the DNA-binding activities of the proteins encoded by the cloned cDNAs.