I am purifying a small heat shock protein that is highly prone to aggregation. To prevent aggregation during purification, I plan to include 200 mM arginine in my buffer. However, for the final elution step, I want to remove arginine so that the eluate is arginine-free. My concern is: once arginine is removed, will the protein re-aggregate?”
How Arginine Prevents Aggregation
Arginine (Arg) → Protein Interactions:
Protein-Protein Aggregation: [Protein] ⟷ [Protein] → [Aggregate] With Arginine: [Protein] + [Arg] ⟷ [Protein-Arg] (Stable) [Arg] blocks hydrophobic patches and reduces protein-protein interactions
The possibilities of re-aggregation depend on the following:
So, yes — aggregation is a real risk when arginine is removed, especially for aggregation-prone proteins.
⚖️Risk Assessment
🔴 HIGH RISK Scenarios
- High protein concentration (>1 mg/mL)
- Rapid arginine removal (direct dialysis)
- Suboptimal buffer conditions (wrong pH, ionic strength)
- Temperature stress during removal
- Aggregation-prone proteins (like heat shock proteins)
🟢 LOW RISK Scenarios
- Low protein concentration (
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