Fig. 5. Detection and purification of specific DNA-binding activities directed against the 30-mer. (A) Gel retardation assay using the 30-mer as probe. A partially purified nuclear extract from slug stage cells was bound to radioactively labelled, 30-mer oligonucleotide (Fig. 2A) with: (1) no competitor (0); (2) the 30-mer wild-type (wt); (3) three mutant forms of the 30-mer (mut 1-3, Fig. 4A and as indicated); or (4) a wild type and a mutant form of a G box oligonucleotide. The major complex (indicated by an arrow) is efficiently competed by the wild-type oligonucleotide but not by the three mutant forms or the G boxes. There are also two minor complexes (indicated by arrowheads) that show the same behaviour as the major complex. (B) Purification of proteins that bind to a 22-mer DNA affinity column. Slug nuclear extracts were purified as shown schematically and the twice affinity-purified proteins were separated by SDS gel electrophoresis. The bands indicated by letters were excised and the proteins identified by mass spectrometry.