Chemiluminescent emsa kit

Electrophoretic mobility shift assays (EMSAs) were performed using the LightShift^TM optimization and control kit (Thermo Scientific) according to the manufacturer’s protocol and as previously described (Batte et al., 2016 (link); Pandey et al., 2019 (link)). Briefly, the binding reaction mixture was prepared with ultrapure water containing 1× binding buffer, 50 ng poly (dI-dC), 2.5% (v/v) glycerol, 0.05% (v/v) NP-40, and 5 mM MgCl₂. Biotin-labeled single-stranded DNA (ssDNA) upstream of the transcription initiation site of the ndh₂ gene and double-stranded DNA (dsDNA) of the ccpE gene in the appropriate concentrations were incubated with increasing concentrations of purified recombinant MsaB–His protein in the binding reaction buffer and unlabeled specific probe when required. The mixture was then incubated at room temperature for 25 min and subjected to electrophoresis at 100 V for 1 h in a pre-run 5% Tris-borate EDTA (TBE) gel. The samples were then transferred to a nylon membrane, incubated for 45 min in the cold, crosslinked, and processed for the detection of samples. The protein–DNA complexes in the gel were then visualized using the Chemiluminescent EMSA kit (Thermo Scientific) according to the manufacturer’s protocol and imaged with a ChemiDoc system (Bio-Rad). The probes used for EMSA studies are listed in Supplementary Table 2.

EMSA was performed using Chemiluminescent EMSA Kit (Thermo) according to manufacturer instructions. Briefly, biotin-labeled oligonucleotides were mixed with indicated proteins in EMSA binding buffer at room temperature for 20 minutes. Samples were subjected to 5% polyacrylamide gel electrophoresis and transferred to nylon membrane. Crosslinked nylon membrane was subjected to an appropriately equipped CCD camera.

The MabHLH28 (N1–309 aa), MaWRKY49C (N101–300 aa), and MaWRKY111 (N1–286 aa) coding regions were subcloned into pGEX-4 T-1 to generate and purify the recombinant GST tag proteins in accordance with the standard protocols (Clontech, Mountain View, CA, USA). The probes containing the MabHLH28-binding sites were biotinylated using a DNA 3′ End Biotinylation Kit (Thermo Scientific, Waltham, MA, USA). EMSA was conducted using a Chemiluminescent EMSA kit (Thermo Scientific, Waltham, MA, USA) as per the manufacturer’s guidelines.

We followed the method of [32 (link)]. EMSA was analyzed using a chemiluminescent EMSA kit according to the manufacturer's protocol (Thermo Scientific, IL, USA). The biotin upper and lower strands of the Stat3 probe (5′-CTTCATTTCCCGGAAATCCCTA-Biotin-3′ and 5′-TAGGGATTTCCGGGAAATGAAG-Biotin-3′) were synthesized. Nuclear extracts were isolated from two breast cancer cells as described previously [35 (link)]. The biotin-labeled probes were incubated with DHTA-treated nuclear proteins in a final volume of 20 μL EMSA buffer containing poly[dI-dC] for 30 min. The mixtures were electrophoresed on a 6% native PAGE gel in 0.5x TBE at 4°C and visualized using a chemiluminescent nucleic acid detection kit (Thermo Scientific, Waltham, MA USA).