Electrophoretic mobility shift assay (EMSA) to show in vitro sequence-specific binding of proteins to the PCS.
The control lane displays EMSA bands obtained by incubating 5 μg nuclear extract with 2 ng 32P-labeled double-stranded PCS (oligo 1). In oligonucleotide competition assays, the32P-labeled double-stranded PCS of the β-promoter (oligo 1) was mixed with various amounts (molar excesses) of nonradioactive PCS-DNA as indicated at the top of the figure. A double-stranded oligonucleotide from −142 to −171 bp relative to the transcription start site of the β-promoter was used as a nonspecific (NS) oligonucleotide (oligo 25 and 26). The band between band 4 and 5 inconsistently appears on the gel. The EMSA bands are numbered and indicated by arrows.
