1. Column Packing

Proper packing is crucial for achieving good resolution and reproducibility. Follow these steps:

• Hydrate the Sephadex G-200: Suspend the dry Sephadex in distilled water at least 12–24 hours at room temperature) to allow full swelling.
• Degas the slurry: Remove air bubbles by gently stirring under vacuum.
• Pour the slurry carefully: Keep the column vertical and fill it gradually while avoiding turbulence.
• Gravity or low-pressure packing: Let the beads settle naturally under gravity or apply low pressure (~1 cm/min flow rate). Avoid compression.
• Top up the column: Once settled, remove excess buffer above the resin and adjust to the correct height.

2. Column Equilibration and sample injection

• Choose running buffer: Since your sample has a pH of ~7, use a buffer that maintains stability, such as PBS (10-50 mM phosphate, 150 mM NaCl, pH 7.4) or Tris-HCl (50 mM, 150 mM NaCl, pH 7.5).
• 2 CV to 3 CV to equilibrate column: Pass at least 2–3 column volumes of equilibration buffer through the column before sample injection.

Al last, I have two suggestions for you: 1. If your sample is a bacterial culture supernatant, why not first perform preliminary purification using an ion exchange column or affinity chromatography before gel filtration? These methods can help remove unwanted contaminants and concentrate your target protein, improving the resolution and efficiency of your gel filtration step. 2. Gel filtration column separates proteins based on their size, relying on differential penetration of molecules into the porous beads. Since separation is purely size-dependent, specific binding interactions are not involved, meaning loading and elution buffers are not required—only an appropriate running buffer for mobilization.