Abstract
This chapter describes the preparation and use of DNA affinity beads that consist of multimerized synthetic oligonucleotides covalently coupled to latex beads. The beads have several advantages for purification of sequence- specific DNA-binding proteins. First, the beads are able to immobilize relatively large amounts of DNA because beads with an extremely small diameter of 0.2μm on average have a larger surface area for total DNA coupling than agarose beads. Second, DNAs coupled on the beads are more accessible to DNA-binding proteins because the beads are free to move in the binding mixture. Third, the property of the bead with a hydrophilic surface lowers nonspecific proteins binding on it. These advantages enable one to directly purify sequence-specific DNA-binding proteins from crude cell extracts within a few hours. This particular method has been successfully employed in the purification of several transcription factors.
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