Akta purifier fplc system

Recombinant EphA2-Sam and Ship2-Sam were expressed in E. coli. Protein constructs along with expression and purification methodologies have been reported in our previous publications^{11 (link),20 (link),23 (link)}. Proteins were either produced in LB (Luria-Bertani) broth or M9 minimal medium supplemented with 1 g/L of ¹⁵NH₄Cl. Bacteria were grown at 37 °C till a cell optical density OD_600nm equal to 0.6, then in the induction step isopropyl β-D-1-thiogalactopyranoside (IPTG) at 1 mM concentration (25 °C, overnight) was used. Purification of his-tagged Ship2-Sam and EphA2-Sam was achieved by affinity chromatography with an Akta Purifier FPLC System (GE Healthcare, Milano, Italy) and a Nickel column. Finally, proteins were dialyzed in PBS pH = 7.4.

Size-exclusion chromatography was carried out at 20 °C on a Superdex 200 10/300 Gl column pre-equilibrated with 20 mM Hepes pH 7.5, using an AKTA Purifier FPLC system (GE Healthcare). Samples (1 ml) were loaded onto the column at 1 mg/ml. Vitamin B12 (1350 Da), horse myoglobin (17,000 Da), chicken ovalbumin (44,000 Da), bovine γ-globulin (15,8000 Da), and bovine thyroglobulin (670,000 Da) (Bio-Rad) were used as MW standards. The logarithm of the MW of the standards were plotted against the ratios of the respective elution volumes (v_e) to the void volume (v₀). The points were then fitted to a linear regression, and the values of slope and intercept were used to determine the molecular weight of SaTrmK in solution.

For purification from inclusion bodies, His-VP1-pET28b construct was transformed in E.coli strain BL21 (DE3), and protein overexpression was carried out at 37 °C for 4 h induced with 1 mM IPTG. Post induction, cells were harvested by centrifugation at 6000 rpm for 20 min at 4 °C and resuspended in 20 mM Tris–HCl pH 8.0. Cell lysis was carried out by sonication, and the resultant pellet was resuspended in a denaturing buffer containing 10 mM PBS, pH 7.4 and 8 M urea to solubilize the inclusion bodies. The urea-solubilized denatured protein was allowed to bind to Ni-NTA agarose beads, followed by washes in denaturing buffer containing 20 mM imidazole, and elution in the same buffer containing 350 mM imidazole.
The eluted protein was dialyzed against buffer solutions containing 10 mM PBS, pH 7.4 with gradually decreasing concentrations of urea, at 4 °C with gentle stirring. Post dialysis, the solution was centrifuged at 12,000g for 10 min at 4 °C to remove any precipitated protein. The cleared supernatant was loaded on a Superdex 200 column (10/300) fitted to an AKTA purifier FPLC system (GE Healthcare) equilibrated in the same buffer for further purification by size exclusion chromatography. Fractions were collected and analyzed on SDS-PAGE.

Flag-HA sequential immunoaffinity purification was carried out as previously described [68 (link)], from 20 mg of nuclear lysates of K562 (MOCK, NPM and NPM-MLF1) cells. Immunoprecipitated proteins were eluted from Flag or HA beads with 4 μg/ml of Flag or HA polypeptides (sequences: Flag: DYKDDDDK or HA:YPYDVPDYA from BioBasic) and molecular size chromatography was performed on the immune-affinity purified complexes with the AKTA purifier FPLC system (GE Healthcare) using Superose 6 increase 10/300 GL column (GE Healthcare). The column was calibrated with Dextran (2000 kDa), Thyroglobulin (669 kDa), Aldolase (158 kDa) and Bovine Serum Albumin (67 kDa) in FPLC buffer (50 mM Tris pH 7.3, 10% Glycerol, 60 mM KCl, 0.2 mM EDTA pH 8.5 mM MgCl₂, 1% NP40 and 0.5 mM DTT). 500 μL of nuclear lysate or Tap-Tag purified sample was loaded on the column. After 0.2 column volume of isocratic elution, 0.5 mL fractions were collected, and proteins were precipitated with 15% TCA. Immunopurified samples or fractions, were first analyzed by silver staining, and then LC-MS/MS analysis was performed to identify interacting partners (Taplin Mass Spectrometry Facility, Harvard University, Boston).
Western blots were performed according to standard protocols. The antibodies used are listed in the S1 Table. Images were acquired with Image Quant LAS400 device (GE Healthcare Life Sciences).