Purpose
Monomer/aggregate call and an orthogonal size check against the designed length.
Setup
- Column: Superdex 75 Increase 10/300 GL — resolves 3-70 kDa, the right range for a 55-65 aa monomer (~6-8 kDa) while still cleanly separating a dimer or small aggregate from it
- Running buffer: PBS pH 7.4 (or 20 mM HEPES pH 7.4, 150 mM NaCl if the prep needs to avoid phosphate)
- Flow rate 0.5 mL/min, 20-50 µL injection of ~1-5 mg/mL protein
- Calibrate the column against a low-MW standard set before trusting absolute apparent MW from retention volume alone
Monomer call
A single, symmetric peak at the retention volume expected for the designed length, with no shoulder toward the void and none toward a dimer-sized peak, counts as monomeric. monomer_pct is that peak's area over total peak area (excluding the void).
If it fails
A void-volume peak or a broad, right-shifted profile means aggregation, not folding failure per se — check Protocol/nanoDSF on the same prep before reworking the design (a normal Tm with a bad SEC trace usually means surface-driven self-association, not misfolding). If it repeats, consider a redesign (SolubleMPNN, lower surface hydrophobicity target) rather than re-purifying the same sequence a second way.