Hybond n positively charged nylon membrane

The upstream regions of the toxP, cbuA0029, and groEL genes were first selected for PCR amplification. PCR products (1 μg) of desired templates were 3′ end‐labeled using a Pierce biotin 3′ End DNA Labeling Kit (Thermo Scientific). The resulting probe reaction mixtures were electrophoresed on a 0.8% agarose gel for 30 min at 100 V and then gel purified with a NucleoSpin Gel and PCR Clean‐up kit (Takara Bio USA). The EMSA binding reaction, consisting of 2.5% glycerol, 5 mM MgCl2, 50 mM KCl, 1 nM biotin‐labeled DNA, and varying concentrations of either ToxP or AntitoxP/ToxP in 1X Binding Buffer (LightShift Chemiluminescence kit; Thermo Scientific), was assembled and incubated at room temperature for 30 min. A nondenaturing loading dye (0.25% bromophenol blue) was added, and the resulting RNA mixtures were resolved on a 10% polyacrylamide gel for 2 h at 100 V. DNA/protein complexes were transferred to a Hybond‐N+ positively charged nylon membrane (Amersham Pharmacia Biotech) using an electroblot transfer system (Bio‐Rad) and cross‐linked with short‐wave UV light in a GS gene linker UV chamber (Bio‐Rad). A North2South chemiluminescence hybridization and detection kit (Thermo Scientific) was used to detect resulting bands. The blot was imaged on a UVP ChemStudio PLUS Imager (Analytik Jena).

Diluted dsRNA (E200 synthesized as described previously [21 (link)]) or purified p14 were applied to a dry Hybond-N+ positively charged nylon membrane (Amersham) and crosslinked with 100 µJ/cm² UV light using a Stratalinker 1800 (Stratagene). The membrane was blocked in 5% skim milk in TBS for 1 h, incubated with J2 antibody (SCICONS) diluted 1:2000 in 1% BSA in TBS for 2 h, followed by HRP-conjugated anti-mouse secondary antibody (Sigma, St. Louis, MO, USA) diluted 1:5000 in 1% skim milk in TBS for 1 h. An ECL solution of luminol, 4-IPBA and H₂O₂ in 100 mM Tris, as described [22 (link)], was added prior to X-ray film exposures.

Ten micrograms of mouse genomic DNA was digested with NcoI (Fermentas, Lithuania) and separated by gel electrophoresis in 1% agarose gel. DNA fragments were transferred onto Hybond-N+ positively charged nylon membrane (Amersham) according to the manufacturer's recommendations. To prepare the probe, a 1.2-kb DNA fragment corresponding to the genomic region immediately downstream of the Trib3 termination codon was purified and radiolabeled in a random-primed labeling reaction containing 50 µCi [α-³²P]-dCTP (Hartmann Analytic GmbH, Germany) using the DecaLabel DNA labeling kit (Fermentas). Autoradiography of the hybridized probe was performed by storage phosphor imaging on a Typhoon Trio imager (GE Healthcare).