For generation of the labeled probe, 5ʹ-IRDye® 700 labeled oligonucleotides were purchased from IDT with the following sequences: ABE; 5ʹ-CGG TGT TGC ACG CGG *CGG GAC GCT CGC GGT AGT TTT* TTC CCA TGA TCA CG-3ʹ and 5ʹ-CGT GAT CAT GGG AAA *AAA CTA CCG CGA GCG TCC CGC CGC* GTG CAA CAC CG-3ʹ and scrambled control probes; 5ʹGTT TAC TAG GTC GAG GTA CTT CGA CGC GCG CCG TCT GCT AGC GCG GTC TG-3ʹ and 5ʹ-CA GAC CGC GCT AGC AGA CGG CGC GCG TCG AAG TAC CTC GAC CTA GTA AAC3ʹ. The AlpA binding element is indicated by asterisks. The oligonucleotides were annealed by mixing them in equimolar amounts in duplexing buffer (100 mM Potassium Acetate; 30 mM HEPES, pH 7.5) and heating to 100 °C for 5 min in a PCR cycler. The cycler was then turned off and the samples were allowed to cool to room temperature while still inside the block. The annealed product was then diluted with water to 6.25 nM for EMSA experiments.
For EMSAs fluorophore labeled DNA probes at a concentration of 0.3125 nM were incubated for 30 min at 20 °C in 20 µl reaction mix (Licor Odysee EMSA Kit) containing 33.4 mM Tris, 70.2 mM NaCl, 12.5 mM NaOAc, 3.75 mM HEPES, 50 mM KCl, 3.5 mM DTT, 0.25% Tween20 and 0.5 µg sheared salmon sperm DNA (ThermoFisher) with proteins. For resolving the reactions, 4% polyacrylamide gels containing 30% triethylene glycol were cast (For two gels: 2 ml ROTIPHORESE®Gel 30 37.5:1 (Roth), 4.5 ml triethylene glycol (Sigma-Aldrich), 1.5 ml 5x TBE-buffer, 7 ml ddH2O, 15 µl TEMED, 75 µl 10% APS). The gels were preequilibrated for 30 min at 130 V in 0.5x TBE-buffer. Samples with added 10x orange dye were then loaded onto the gel at 4 °C and the voltage set to 300 V until the samples entered the gel completely. The voltage was then turned down to 130 V and the gel was run until the migration front reached the end of the gel. The gels were imaged using the Licor Odyssey imaging system using the 700 nm channel. For generation of the figures, the scanned image was converted to greyscale and brightness and contrast adjusted. The unprocessed scan is available as Supplementary Fig.15 .
For EMSAs fluorophore labeled DNA probes at a concentration of 0.3125 nM were incubated for 30 min at 20 °C in 20 µl reaction mix (Licor Odysee EMSA Kit) containing 33.4 mM Tris, 70.2 mM NaCl, 12.5 mM NaOAc, 3.75 mM HEPES, 50 mM KCl, 3.5 mM DTT, 0.25% Tween20 and 0.5 µg sheared salmon sperm DNA (ThermoFisher) with proteins. For resolving the reactions, 4% polyacrylamide gels containing 30% triethylene glycol were cast (For two gels: 2 ml ROTIPHORESE®Gel 30 37.5:1 (Roth), 4.5 ml triethylene glycol (Sigma-Aldrich), 1.5 ml 5x TBE-buffer, 7 ml ddH2O, 15 µl TEMED, 75 µl 10% APS). The gels were preequilibrated for 30 min at 130 V in 0.5x TBE-buffer. Samples with added 10x orange dye were then loaded onto the gel at 4 °C and the voltage set to 300 V until the samples entered the gel completely. The voltage was then turned down to 130 V and the gel was run until the migration front reached the end of the gel. The gels were imaged using the Licor Odyssey imaging system using the 700 nm channel. For generation of the figures, the scanned image was converted to greyscale and brightness and contrast adjusted. The unprocessed scan is available as Supplementary Fig.
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