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Updated: Jul 21, 2026
Identification of Protein Interacting Partners Using Tandem Affinity Purification
Published on: February 25, 2012
Direct purification of multiple ATF/E4TF3 polypeptides from HeLa cell crude nuclear extracts using DNA affinity latex
Y Inomata1, H Kawaguchi, M Hiramoto
1Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan.
Abstract:
We developed a method using affinity latex particles to rapidly and efficiently purify DNA-binding proteins directly from crude cell extracts. The particles are composed of a styrene core and a polyglycidyl methacrylate surface, to which DNA oligomers were immobilized by means of epoxy groups. Multiple polypeptides were copurified, which bound to the ATF/E4TF3-binding site from crude nuclear extracts of HeLa cells, within a few hours. Affinity-purified polypeptides stimulated transcription in vitro from a promoter in which ATF/E4TF3-binding sites were present. At least eight polypeptides with molecular masses of 116, 80, 65, 60, 55, 47, 45, and 43 kDa were copurified. About 2 micrograms of the 43-kDa protein was purified directly from 8 mg of crude nuclear extracts. All the polypeptides directly bound to the same DNA sequence and were thought to form a family. The results indicated that the particles are useful for quickly purifying various DNA-binding proteins directly from crude cell extracts.
