Novex 4 20 native tris glycine gels

A 57-bp oligonucleotide consisting of the SaPIbov1 P_str promoter region was used in the assay (Supplementary Table S1) [35 (link)]. 25–50 ng of P_str DNA was combined with 1–2 μM Stl both with and without 6–12 μM WT or mutant Dut_NM1, and incubated for 30–60 min at 22 °C in EMSA buffer [34 (link)]. The mixture was separated on Novex™ 4–20% native Tris-Glycine gels (Life Technologies) [21 (link)]. For the nucleotide dependence experiments, 2 mM dUTP or 2 mM dUMP were added to the mixture containing 6–12 μM WT or mutant Dut_NM1 and 1.5 μM Stl and 25 ng P_str DNA, and the assay carried out as above.

To obtain purified Stl protein, the SaPIbov1 Stl coding sequence was
cloned into the vector pGEX-6P-1 in-frame with the GST sequence. The protein was
expressed in BL21 Star (DE3) cells, lysed by addition of lysozyme,
freeze-thawing and sonication, and purified by batch affinity on
glutathione-sepharose beads (Amersham Biosciences). The GST tag was removed by
overnight cleavage with PreScission protease (GE Health Sciences), while on the
beads.
A 170 bp DNA fragment consisting of the SaPIbov1
P_stl–P_strpromoter region was generated by PCR using the primers described by Tormo-Mas et
al. 2010 [20 (link)].
36–75 ng of DNA were combined with 0.3–1.0 μM Stl (final
concentration) and 0–6.4 μM Dut_NM1 and incubated at
22 °C for 1 h before separation on Novex^™4–20 % native Tris-Glycine gels (Life Technologies, Carlsbad,
CA). The gels were stained with 0.15 μg/ml ethidium bromide and
visualized under UV light.