Mini protean tetra cell electrophoresis system

EMSA reactions (15 µl) were performed in binding buffer containing 25 mM Tris-HCl pH 8.0, 2 mM ATP, 2 mM MgCl₂, 1 mM DTT, 50 mM NaCl, 0.1 mg ml⁻¹ BSA (New England Biolabs) and 5′-end-FITC-labelled 25 nM DNA substrate (in molecules). All of the steps except for the assembling reactions and protein addition were performed in the dark. The reactions were assembled on ice and recombinant proteins were added to reactions, mixed and incubated for 10 min at 37 °C in the dark. Reactions were supplemented with 5 µl of EMSA loading buffer (50% glycerol, bromophenol blue) and resolved with 6% native TBE polyacrylamide gel (19:1 acrylamide-bisacrylamide, Bio-Rad) using the Mini-Protean Tetra Cell electrophoresis system (Bio-Rad) at 80 V for 45 min on ice. Finally, gels were imaged using the ChemiDoc MP imaging system. Scans of the gels are provided in the Supplementary Information.

Protein samples were boiled at 95°C for 5 min and separated by SDS-PAGE in 10–15% acrylamide gels using a Mini-PROTEAN tetra cell electrophoresis system (Bio-Rad). Proteins were transferred from gels into 0.2 μm-pore size nitrocellulose membranes (10600001, GE Healthcare) using a Trans-Blot Cell electroblotting system (Bio-Rad). The presence of total protein was detected by Ponceau S solution (P7170, Sigma-Aldrich) staining. Non-specific binding was blocked by incubation of the membranes with 0.1% Tween 20-TBS solution containing 5% skimmed milk powder or BSA (A3912, Sigma-Aldrich) for 1 h at room temperature (RT), prior to addition of the primary antibody (KRT). After washing the unbound primary antibody with 0.1% Tween 20 -TBS three times, membranes were incubated with the corresponding HRP-linked secondary antibody (KRT) for 1 h at RT. Membranes were washed three times to remove the excess of secondary antibody and the Clarity Western ECL substrate (170–5061, Bio-Rad) was subsequently added. The chemiluminescent signal from immunoreactive proteins was detected in the ImageQuant LAS 4000 imaging system (GE Healthcare). Protein bands were quantified by densitometric analysis using the open-source image processing program Fiji software (https://imagej.net/software/fiji/) and normalized to β-actin housekeeping protein expression.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to visualize the total protein content in the extracts, but also as a purification step before LC/MS analysis. Twelve micrograms of protein extract were briefly heated in Laemmli buffer [42 (link)] with dithiothreitol and loaded onto a 15% bisacrylamide gel. Electrophoretic separation was performed at 170 V by use of a Mini-Protean Tetra Cell electrophoresis system (BioRad, Hercules, CA, USA). The separated proteins were visualized by staining with Coomassie brilliant blue G250.
Tropomyosin was excised from the SDS-PAGE gel for mass spectrometric analysis. The SDS-PAGE bands between 34 and 40 kDa (Appendix A) were destained and digested with trypsin, according to the method designed by Abel Rahman et al. [26 (link)], before injection into the LC/MS system. Tropomyosin quantification was based on IVELEEELR (sequence) peptide determination, as suggested by Wang et al. [37 (link)].

Hand cast tris–glycine-SDS gels were used for protein electrophoresis in tris–glycine running buffer (25 mM tris pH 8.5, 190 mM glycine, 0.1% SDS) using the Mini-PROTEAN^® Tetra cell electrophoresis system (Bio-Rad). Samples were diluted with 4x Laemmli buffer (0.25 M Tris pH 6.8, 40% (v/v) glycerol, 20% (v/v) 2-mercaptoethanol, 8% sodium dodecyl sulfate, 0.04% bromophenol blue) and water, then heated to 85 °C for 5 min prior to loading on gels.

The electrophoretic mobility of conjugates labeled with fluorescein was studied by polyacrylamide gel electrophoresis. Continuous native PAGE under acidic or basic conditions was performed with vertical Mini-PROTEAN Tetra Cell electrophoresis system (Bio-Rad, Hercules, CA, USA) using Minigels (100 mm × 8 mm × 1 mm). Two types of buffers were used: 80 mM β-alanine-acetate, pH 5.0, and 50 mM Tris-acetate, pH 7.2. 2 µµL of a sample containing 1 µL dendrimers (10 µg) and 1 µL charge buffer (30% glycerol, 8 mM β-alanine-acetate, pH 5.0, or 5 mM Tris-acetate, pH 7.2) was applied to 4% gel. The electrophoresis was performed at 200 V at room temperature for about 30 min under acidic conditions and 20 min under alkaline conditions.