Biorad mini protean gel system

EMSA [37 (link)] was performed with the BioRad Mini Protean gel system (BioRad, USA) at 90V for 1 hour. The binding reactions were performed for 30 minutes with the Odyssey™ EMSA Buffer Kit (LI-COR, USA) according to the manufacturer recommendations with some modifications. Binding reaction: 1x binding buffer, 2.5mM DTT/0.25% Tween20, 2.5% glycerol, 8ng/μl of SPIB protein, 250nM of probe and a total incubation volume of 20 μl. Products were resolved by polyacrylamide gel electrophoresis using a 10% Mini-PROTEAN^® TBE Precast Gel (BioRad), and 0.5 × TBE buffer, then analyzed by staining with GelRed Nucleic Acid Gel Stain, (Biotium, USA) and visualized by UV lamp. Purified SPIB (Human) Recombinant Protein (P01) was purchased from Abnova (Taiwan). Double stranded (ds) 50bp DNA probes were annealed from ssDNA oligonucleotides in 1xTE buffer pH 8 with addition of 50mM NaCl, final probe concentration was 1.5 μM. Probes were heated to 90^oC for 5 min, then slowly cooled down to RT in 2h. The 10% TBE gels were prerun in 0.5x TBE buffer for 1h at 90V. Specificity of binding was demonstrated by mutation of the putative SPIB core binding motif in two probes, GG was changed to TT (GGAA→TTAA).

Samples were separated by electrophoresis on 12% polyacrylamide gels using a Bio-Rad Mini Protean Gel system (Bio-Rad Laboratories, Watford, United Kingdom). For reducing, denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), samples were boiled with Laemmli buffer containing β-mercaptoethanol at 95 °C for 5 min (crude cellular lysates and medium samples) or 10 min at 50 °C (fractions from ion exchange chromatography). For native analysis, samples were not boiled and no sodium dodecyl sulfate nor β-mercaptoethanol was used in samples, gels or running buffer. The polyacrylamide gels were subsequently immunoblotted using antibodies against the C-terminal HA-tag (Sigma-Aldrich, Dorset, United Kingdom) and against the C-terminus of the IF protein (abcam) to detect the protein of interest.