Sybr green emsa nucleic acid gel stain

Atu1419 or Atu1419-H3A mutant were mixed with different promoter regions (Patu1416–1417, Patu1418–1419, PhcaR, Patu1420 and PvirB) and with variants of the atu1418–1419 and atu1420 regions. The intergenic regions of virB was used as a nonspecific control probe. Gel-shift assays/EMSA were performed in 10 μl reaction mixture containing 30 nM of DNA probe without and with Atu1419 at different concentrations in 50 mM Tris–HCl pH 8 and 150 mM NaCl. 50 to 300 μM of MEF, MH4F, H4F or citrate were added for testing their influence on Atu1419–DNA complex formation. After incubation for 30 min at room temperature, the samples were separated by electrophoresis in TBE buffer (45 mM Tris–HCl pH 8, 45 mM boric acid and 1 mM EDTA) on non-denaturing 6% or 12% polyacrylamide gels at 150 V and 4°C for 2 h. Gels were then stained with either SYBR® Green EMSA nucleic acid gel stain (Invitrogen, Carlsbad, CA, USA) or ethidium bromide for 20 min. DNA was visualized under UV light (Fisher Bioblock Scientific, Illkirch, France or UVP BioDoc-it2 Imager, Analytic Jena, Germany).

OmpR binding studies were performed using purified N-terminal His-tagged OmpR protein and DNA fragments containing the acrR and acrAB control regions. The acrR promoter fragment p222 used in the transcriptional fusion with gfp was employed in the EMSA. However, a new acrAB promoter fragment p225 was amplified for this purpose by PCR with primer pair FacAB1/RacAB225 (S1 Table). OmpR-His₆ synthesized in E. coli M15 was purified using Ni²⁺-NTA agarose as described previously [30 (link)]. The EMSA method was adapted from a previously published protocol [31 (link)]. For phosphorylation of OmpR, 20 mM acetyl phosphate (Sigma) was used. To confirm binding specificity, a 304-bp fragment of Y. enterocolitica Ye9 16S rDNA generated by PCR using primer pair 16SR1/16SR304 (S1 Table) was included as a non-specific competitor in all binding reactions. The reaction mixtures were analyzed by electrophoresis on 6% non-denaturing polyacrylamide gels run in TBE buffer (Tris-borate-EDTA) and DNA bands were stained with SYBR Green EMSA nucleic acid gel stain (Invitrogen).

RNA binding was studied by means of the electrophoretic mobility shift assay (EMSA) with minor modifications. Briefly, 0.5 μg/ml of HCV-NS5BΔ21 was incubated with 1 μg of a 500 bp RNA at 37°C for 30 min in 20 μl of a binding buffer containing 10 mM Tris pH 7.5, 1 mM EDTA, 100 mM KCl, 0.1 mM DTT, 5% vol/vol glycerol, 10 μg/ml bovine serum albumin (BSA), in the presence of increasing concentrations of the tested compound. Samples were then loaded onto a non-denaturing agarose gel. The gel was incubated 20 min in SYBR green EMSA Nucleic Acid Gel Stain (Invitrogen, Carlsbad, CA, USA). After washing, the gel was visualized under ultraviolet transillumination.