Anion-Exchange Chromatography-Based Protein Purification: A Separation Technique to Isolate a Protein of Interest From Dialyzed Bacterial Lysate Based on Net Charge

1. Protein Purification

1. Anion-exchange chromatography
   1. Dialysis
      1. Prepare anion-exchange chromatography (AIEX) buffer A by dissolving 20 mM Tris, 1 mM EDTA and 1 mM ethylene glycol-bis(2-aminoethylether)-N, N, N', N'-tetraacetic acid (EGTA) in deionized water and adjust the pH to 8.5 with HCl. For dialysis prepare 2 x5 L and for chromatography 2x 1 L of AIEX buffer A.
         NOTE: The dialysate volume should be at about 100 times the sample volume. All buffers used for chromatography should be filtered (0.45 μm or smaller) and degassed (e.g., by ultrasonic bath or vacuum degassing).
      2. Cut dialysis tubing (molecular weight cut-off [MWCO]: 3.5 kDa) into an appropriate length with additional space for air to ensure sample buoyancy above the rotating stir bar.
         NOTE: Glycerol preserves the membrane and must be removed before use.
      3. To reduce the viscosity of the cleared protein solution, dilute the solution with 25 mL of AIEX buffer A to facilitate subsequent application to the chromatography column. Attach the first closure onto the tubing, load the sample into the membrane and attach the second closure at least 1 cm from the top end of the tubing.
      4. Place the 5 L container with AIEX buffer A on a stir plate, add a stir bar and the membrane filled with protein solution. Adjust the speed to rotate the sample by avoiding interference with the rotating stir-bar. Dialyze for 12−24 h at 4 °C, then replace the dialysate buffer (AIEX buffer A) by a fresh pre-cooled preparation and continue for at least 4 additional hours. Transfer the dialyzed protein solution to a 50 mL tube and filter through a 0.45 μm filter unit.
         NOTE: Storage possible.
   2. Chromatography
      1. Start the liquid chromatography system (FPLC) with general maintenance, connect column buffers AIEX A and AIEX B (AIEX buffer A with 1 M NaCl) and the anion-exchange resin containing column. Refer to Table 1 for general chromatographic parameters.
         NOTE: Buffers, column and FPLC equipment should be equilibrated to the same temperature before starting the run (refer to chromatographic parameters in Table 1, Table 2).
      2. Equilibrate the column with AIEX buffer A, subsequently load the sample onto the column and elute the proteins with a linear gradient from 0% to 100% high-salt buffer (AIEX B). Refer to Table 2 for a detailed method protocol.
      3. Collect 2 mL fractions during elution and analyze 10 μL of each fraction on a Coomassie-stained 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Pool the fractions containing S100A12 protein for dialysis.
         NOTE: The molecular weight of S100A12 is 10,575 Da.