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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.
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