Ni nta slurry

Proteins were expressed from the plasmid pQE32 containing lytB or lytC in E. coli BL21 (DE3). Cells were grown in LB supplemented with ampicillin (50 mg ml⁻¹) up to OD₆₀₀ 0.5 and induced by the addition of 0.5 mM IPTG for 16 h at 16 °C. Purified proteins were extracted according to Qiagen protocol (The QIAexpressionist). Cells were harvested by centrifugation and resuspended in lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole at pH 8.0). Next, lysozyme (1 mg ml⁻¹), RNaseA (10 μg ml⁻¹), DNase I (5 μg ml⁻¹) were added and cells were incubated in ice for 30 min and treated using Fastprep (FastPrep (MP) 6.5, 30 s, ×3) and the lysates were collected after centrifugation at 10,000 × g for 20 min. The lysates were then mixed with Ni-NTA slurry (Qiagen) and incubated at 4 °C for 1 h with gentle mixing. The mixture was then poured into Poly-Prep chromatography column (Bio-Rad) and washed three times with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) and eluted in elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0). The eluted fractions were pooled and used for investigation.

Three independent biological replicates were performed. Cells in 10 cm dishes were treated with or without ADI-PEG20 (10 µM) for 72 h, washed with PBS and lysed in 1× Cell Signaling Cell Lysis Buffer plus 1 mM phenylmethylsulfonyl fluoride. Lysates were sonicated in a water bath on ice for 15 s and insoluble material was removed with a 14,000×g centrifugation for 10 min at 4 °C. Five 200 µg aliquots of lysate were made for each sample. Each aliquot was desalted with 600 µL GE 2-D Clean-Up kit and processed through to trypsin digest as in ABPP. Lysates were desalted using Oasis HLB columns per manufacturer’s instructions. Samples were then diluted with 1 mL 90% ACN (acetonitrile), and phosphopeptides were enriched with 20 µL of Qiagen Ni-NTA slurry for 30 min at 25 °C with end-over-end rotation. Beads were washed four times with 1 mL 80% ACN, 0.1% trifluoroacetic acid and eluted with 250 µL 50% ACN, 2.5% ammonia, and 2 mM phosphate buffer pH 10. Lysates were acidified to pH <3 with formic acid, vacuum concentrated to dryness, desalted with a C18 ziptip using manufacturer’s instructions, vacuum concentrated to dryness, resuspended in 0.5% formic acid and analyzed by LC–MS.

MITF DNA-binding domains (residues 180–296) that were either WT, K206Q or K206R were expressed in Escherichia coli BL21(DE3) cells (Agilent Technologies). Cultures were grown in Luria-Bertani broth to an OD₆₀₀ of 0.7–0.8. Recombinant protein overexpression was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG; 0.5 mM final concentration) and the cultures were incubated for a further 6 h. Cells were harvested by centrifugation, washed in PBS and frozen on dry ice. After thawing, cells were suspended in lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, 10% v/v glycerol, pH 7.4 [NaOH], 20 mg/ml lysozyme [Invitrogen], 1× protease inhibitor cocktail [Roche]). Cells were lysed by sonication and centrifuged. Clarified lysate was mixed by rotation with a 50% Ni-NTA slurry (QIAGEN) previously equilibrated in lysis buffer and loaded in gravity flow columns (Bio-Rad). After extensive washing in wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, 10% v/v glycerol, pH 7.4 [NaOH]), bound material was serially eluted in elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, 10% v/v glycerol, pH 7.4 [NaOH]). Fractions were analysed by SDS-PAGE and Coomassie staining to determine purity, and pure fractions were pooled and glycerol added to a final concentration of 30% v/v.

MBP- and GST-tagged proteins were both expressed in E. coli strain BL21 (DE3) (plyS). Cells were harvested by centrifugation and resuspended in 20 ml of lysis buffer (50 mM Tris-HCl (pH 8.0) 500 mM NaCl, 0.22 mg/ml lysozyme, 100 μM PMSF, and 10 mM DTT) and sonicated on ice. The lysate was centrifuged, and the supernatant was mixed with Ni-NTA slurry (Qiagen, Venlo, the Netherlands) and rocked for 60 min at 4 °C. The mixture was poured through a Ni-NTA column and washed with 50 ml of phosphate wash buffer (50 mM Tris-HCl (pH 8.0), 20 mM imidazole, 500 mM NaCl, and 10% glycerol). Purified protein was eluted with elution buffer (500 mM imidazole, 1 M NaCl, 10% glycerol, and 50 mM Tris-HCl (pH 8.0)). The imidazole and excess NaCl were removed by dialysis in buffer (50 mM Tris-HCl, 150 mM NaCl and 10% glycerol).
MBP and MBP-EZH2 proteins were incubated at 4 °C with GST-Ku80 proteins overnight. MBP-tagged proteins were captured by incubation with anti-MBP magnetic beads (New England Biolabs, Ipswich, MA, USA) at 4 °C for 3 h. The bead pellet was washed five times with 500 μl of buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 10% glycerol and 2 mM EDTA). Samples were boiled and subjected to SDS-PAGE.