Superdex 75 prepgrade column

Expression and purification were performed as previously described [19, 20]. Briefly, the pET5α/αSynuclein (136 TAT) plasmid (wt‐plasmid by Philipp Kahle, LMU Munich; 136‐TAC/TAT‐mutation by Matthias Habeck) was transformed into BL21(DE3) Escherichia coli (New England Biolabs, Frankfurt am Main, Germany). Protein expression was induced with 1 m IPTG (Peqlab, Erlangen, Germany) for 4 h at 37 °C. Cells were lysed by boiling after heat inactivation of proteases. After centrifugation, the supernatant was filtered through a Filtropur S 0.2 filter (Sarstedt, Nümbrecht, Germany), loaded onto a 5‐mL HiTrap Q HP anion exchange column (GE Healthcare, Munich, Germany) and eluted over a linear NaCl gradient (25–500 mm). The fractions containing aSyn were pooled, concentrated and gel filtrated via a Superdex 75 prep grade column (25 mL; GE Healthcare). After adjusting the protein concentration to 1 mg·mL⁻¹, aliquots were frozen in liquid nitrogen and stored at −80 °C until use.

BL21(DE3) cells containing GST-Nck1 (or Nck variant, both His- and non-His-tagged) were collected by centrifugation and lysed by sonication in 25 mM Tris-HCl (pH 8.0), 200 mM NaCl, 2 mM EDTA (pH 8.0), 1 mM DTT, 1 mM PMSF, 1 μg/ml antipain, 1 μg/ml pepstatin, and 1 μg/ml leupeptin. Centrifuge-cleared lysate was applied to Glutathione Sepharose 4B (GE Healthcare) and washed with 25 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 1 mM DTT. GST was cleaved from protein by TEV protease treatment for 16 hr at 4°C. Cleaved protein was applied to a Source 15 Q anion exchange column and eluted with a gradient of 0 → 200 mM NaCl in 20 mM imidazole (pH 7.0) and 1 mM DTT. Eluted protein was pooled and applied to a Source 15 s cation exchange column and eluted with a gradient of 0 → 200 mM NaCl in 20 mM imidazole (pH 7.0) and 1 mM DTT. Eluted protein was concentrated using Amicon Ultra 10K concentrators and further purified by size exclusion chromatography using a Superdex 75 prepgrade column (GE Healthcare) in 25 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM βME, and 10% glycerol.

BL21(DE3) cells expressing GST-Nck were collected by centrifugation and lysed by sonication in 25 mM Tris-HCl (pH 8.0), 200 mM NaCl, 2 mM EDTA (pH 8.0), 1 mM DTT, 1 mM PMSF, 1 μg/ml antipain, 1 μg/ml benzamidine, 1 μg/ml leupeptin, and 1 μg/ml pepstatin. Centrifugation-cleared lysate was applied to Glutathione Sepharose 4B (GE Healthcare) and washed with 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 1 mM DTT. GST was cleaved from protein by TEV protease treatment for 16 hr at 4 °C. Cleaved protein was applied to a Source 15 Q anion exchange column and eluted with a gradient of 5 → 250 mM NaCl in 20 mM imidazole (pH 7.0) and 1 mM DTT. Eluted protein was pooled and applied to a Source 15 S cation exchange column and eluted with a gradient of 0 → 500 mM NaCl in 20 mM imidazole (pH 7.0) and 1 mM DTT. Eluted protein was concentrated using Amicon Ultra 10 k concentrators and further purified by size exclusion chromatography to remove any protein aggregates using a Superdex 75 prepgrade column (GE Healthcare) in 25 mM HEPES (pH 7.5), 150 mM NaCl, and 1 mM βME.

BL21(DE3) cells expressing GST-Nck were collected by centrifugation and lysed by sonication in 25 mM Tris-HCl (pH 8.0), 200 mM NaCl, 2 mM EDTA (pH 8.0), 1 mM DTT, 1 mM PMSF, 1 μg/ml antipain, 1 μg/ml benzamidine, 1 μg/ml leupeptin, and 1 μg/ml pepstatin. Proteins were affinity-purified with Glutathione Sepharose 4B (GE Healthcare). GST was cleaved from protein by TEV protease treatment for 16 hours at 4 °C, followed by anion exchange chromatography using Source 15 Q resin. Eluted protein was pooled and purified further by a Source 15 S cation exchange column. Eluted protein was concentrated using Amicon Ultra Centrifugal Filter units (Millipore) and further purified by size exclusion chromatography using a Superdex 75 prepgrade column (GE Healthcare) in 25 mM HEPES (pH 7.5), 150 mM NaCl, and 1 mM DTT.

The vectors used in this work were prepared and expressed as described previously (29 (link)). All chemicals used for protein preparation and fluorescence measurements were of ultrapure grade. For each experiment, proteins were thawed on wet ice and centrifuged at 15,000 × g for 10 min at 4 °C, and then protein concentrations were measured based on the absorbance at 280 nm using extinction coefficients of 17,420, 15,930, and 17,285 m⁻¹ cm⁻¹ for p53C, p63C, and p73C, respectively. Soluble fractions obtained after cell lysis in 50 mm Tris-HCl, pH 7.4, containing 150 mm NaCl, 5 mm DTT, and centrifugation at 18,000 × g for 15 min at 4 °C were loaded onto nickel-nitrilotriacetic acid resin (Qiagen) followed by gel filtration chromatography using a Superdex 75 Prepgrade column (GE Life Sciences, catalog no. 17-5174-01). Protein elution was monitored using an Äkta prime system at 280 nm, and purity was checked by 15% SDS-PAGE. The purified proteins were concentrated as required using Amicon Ultra-15 10K filter devices (Millipore, catalog no. UFC901024). p63C and p73C were stored at −80 °C, and 5% glycerol was added to p53C and p53C QM before storage in liquid nitrogen.