Gel-mobility shift assays reveal a distinct, labile activity that binds to the gyrB promoter. (A) A 500 base pair [32P]-end-labeled DNA fragment containing the gyrB promoter was used as a binding template for incubation with increasing amounts of fresh C. crescentus crude cell extract (approximately 1 to 3 mg/ml), without (lanes 1–4) or with (lanes 5–7) increasing amounts of anti-RNAP antibody. (B) The same binding template was incubated with 3 μl of fresh crude extract (lanes 4–5) or extract that had been previously re-frozen (lanes 2–3), with and without anti-RNAP antibody (3 μl). All binding mixtures were incubated at room temperature for 10 minutes and immediately loaded onto native 6% polyacrylamide gels. Black solid arrows indicate probe alone; gray solid arrows indicate a potential freeze-thaw-labile, shifted species; white, broken arrows indicate a binding activity that is super-shifted by anti-RNAP antibody.