36] Magnetic DNA affinity purification of yeast transcription factor

Abstract

This chapter describes the preparation of magnetic DNA affinity beads and their use in the purification of sequence-specific DNA-binding proteins. The protein chosen for the illustration of the method is yeast transcription factor z––one of the most complex transcription factors characterized so far. The chapter presents an improved variant of the previously reported magnetic DNA affinity purification method: starting with a partially purified protein fraction (purity < 0.2%), essentially the pure preparations of yeast transcription factor z were obtained in less than 30 min with a single adsorption step. Improved purity and yields were obtained by saturating the DNA affinity beads with the specific DNA binding protein and by including competitor DNA only during the washing of the beads. With the same starting material, this 30-min procedure yielded higher purity than the normal method, which uses columns and requires several days.