Protocol for GST-Tagged Protein Purification

Protocol for GST-Tagged Protein Purification

1. Protein Expression

• Plasmid Transformation: Transform the pGEX-4T1 vector containing GST-tagged EAF1 into E. coli BL21 competent cells through heat shock at 42 °C for 60 seconds.
• Plating: Spread the transformed bacteria onto an LB agar plate containing ampicillin and incubate overnight at 37°C to select for positive transformants.
• Culture Growth:
  • Inoculate a single colony into LB medium containing ampicillin and grow overnight at 37°C with shaking (200 rpm).
  • Dilute the culture 1:100 in fresh LB containing ampicillin and grow at 37°C until OD600 ≈ 0.6–0.8.
• Induction: Add 100 μM IPTG and incubate overnight at 16°C for soluble protein expression.

2. Cell Harvest and Lysis

• Harvesting: Pellet cells by centrifugation at 4,000 × g, 4°C, 20 min. Discard supernatant.
• Resuspension: Resuspend pellet in binding buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.5) supplemented with protease inhibitor.
• Sonication: Lyse cells on ice using repeated cycles of 10-second sonication followed by 10 seconds off, continuing for 30 minutes. Pause the sonication every 10 minutes for a 1-minute cooling interval.
• Clarification: Centrifuge at 12,000 × g, 4°C, 30 min to remove debris. Collect supernatant (soluble fraction).

3. Affinity Purification

• GST Beads Preparation:
  • Equilibrate GST beads in binding buffer.
  • Incubate clarified lysate with beads for 1–2 hours at 4°C with gentle rotation.
• Washing:
  • Pellet beads (500 × g, 2 min), discard flow-through.
  • Wash 3× with 1mL Binding Buffer.
• Elution:
  • Incubate beads with 10 mM reduced glutathione in 50 mM Tris-HCl (pH 8.0) for 10 min at 4°C.
  • Centrifuge and collect eluate. Repeat 2–3 times.

4. Buffer Exchange

• Dialysis:
  • Load the eluted protein into a 10-kDa MWCO dialysis tubing.
  • Dialyze against 1 L of storage buffer (e.g., PBS) at 4 °C with gentle stirring, performing two buffer exchanges during the process.
• Concentration:
  • After dialysis, concentrate the protein using a 10-kDa MWCO centrifugal filter (if needed).
  • Determine protein concentration by Bradford assay or absorbance at 280 nm.

5. Quality Control

• SDS-PAGE: Verify purity by Coomassie staining.
• Western Blot: WB with anti-GST antibody if necessary.
• Storage: Aliquot and store at −80°C.