Tbe polyacrylamide slab gels

Reaction mixtures contained (20 μl): test compound (0 or 10 μM SalA, 0.2 μM Rif, 400 μM Stl, 100 μM CBR703, 20 μM MyxB, or 100 μM Lpm), 40 nM E. coli RNAP holoenzyme, 10 nM DNA fragment containing positions −42 to +426 of the lacUV5(ICAP) promoter (Naryshkin et al., 2001 (link)), and 100 μg/ml heparin in TB. Reaction components other than DNA and heparin were pre-incubated 5 min at 24°C, DNA was added and reaction mixtures were incubated 15 min at 37°C; heparin was added and reactions were incubated 2 min at 37°C to disrupt non-specific RNAP-promoter complexes and RNAP-promoter closed complexes (Cech and McClure, 1980 (link)). Products were applied to 5% TBE polyacrylamide slab gels (Bio-Rad), gels were electrophoresed in TBE, and gels were stained with SYBR Gold Nucleic Acid Gel Stain (Life Technologies, Grand Island, NY).

Reaction mixtures contained (20 μl): test compound (0 or 0.5 μM GE, or 2.2 μM Rif), 40 nM E. coli RNAP holoenzyme, 10 nM DNA fragment containing positions −42 to +426 of the lacUV5(ICAP) promoter (Naryshkin et al., 2001 (link)), and 100 μg/ml heparin, in TB. Reaction components other than DNA and heparin were pre-incubated 10 min at 37°C; DNA was added and reaction mixtures were incubated 15 min at 37°C; heparin was added and reactions were incubated 2 min at 37°C to disrupt non-specific RNAP-promoter complexes and RNAP-promoter closed complexes (Cech and McClure, 1980 (link)). Products were applied to 5% TBE polyacrylamide slab gels (Bio-Rad, Hercules, CA), gels were electrophoresed in TBE (90 mM Tris-borate, pH 8.0, and 2 mM EDTA), and gels were stained with SYBR Gold Nucleic Acid Gel Stain (Life Technologies).