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Since each operator can assume two states (free and taken by a transcription factor), only four possible configurations were possible, and for a unique transcription factor, only four binding reactions were present.
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Binding reactions were scaled up for immunoprecipitation of photoaffinity-labeled subunits.
Binding reactions were the same as in (A).
For supershift assays, binding reactions were set as described above.
Binding reactions were set up with varying protein combinations.
Binding reactions were allowed to take place overnight at 4°C.
Binding reactions were performed in RNA binding buffer (10 mM Tris-HCl (pH 7.4), 10 mM KCl, 1 mM MgCl2).
Binding reactions were carried out in triplicate.
Binding reactions were performed using 200 ng of semipurified protein.
Binding reactions were incubated for 20 min at room temperature.
Binding reactions were performed as previously described [ 13].
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