Protein purification turns a complex cell lysate into a single, usable protein. The strategy depends on the protein’s properties, the downstream application, and the scale you need.
The three-stage strategy
Stage 1: Capture. Quickly concentrate your protein from a complex mixture. Affinity chromatography is the most common choice if your protein has a tag (His, GST, FLAG, MBP). Typically 80–95% pure in one step.
Stage 2: Intermediate purification. Remove remaining contaminants. Ion exchange chromatography is the workhorse — separates by charge. Hydrophobic interaction chromatography (HIC) is an alternative.
Stage 3: Polishing. Size exclusion chromatography (SEC) removes aggregates and gives a final highly pure preparation. Provides built-in QC of oligomeric state.
Affinity tag systems
| Tag | Resin | Elution | Notes |
|---|---|---|---|
| His6/8/10 | Ni-NTA, Co²⁺ resins | Imidazole gradient | Most common; small tag |
| GST | Glutathione resin | Reduced glutathione | Large tag; can dimerize |
| MBP | Amylose resin | Maltose | Large; improves solubility |
| FLAG | Anti-FLAG M2 | Acidic pH or FLAG peptide | Small; excellent specificity |
| Strep-tag II | Strep-Tactin | Desthiobiotin | Small; physiological elution |
Step-by-step workflow (His-tagged protein)
1. Express and harvest
Grow E. coli with your expression construct, induce with IPTG (typical: 0.5 mM at OD₆₀₀ ~0.6, 16–18 °C overnight for soluble protein). Harvest by centrifugation, freeze pellet at -80 °C if not processing immediately.
2. Lyse
Resuspend pellet in lysis buffer (50 mM Tris pH 8, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF). Lyse by sonication on ice. Clarify by centrifugation at 20,000 × g for 30 minutes.
3. Affinity capture
Pass clarified lysate over Ni-NTA column equilibrated in lysis buffer. Wash with 20–40 mM imidazole. Elute with 250–500 mM imidazole.
4. Tag removal (optional)
Cleave with TEV, PreScission, or thrombin overnight at 4 °C, then run the digest over a fresh Ni-NTA column to remove the cleaved tag and protease.
5. Polish by SEC
Concentrate the cleaved protein and load on a size exclusion column (Superdex 75 for <70 kDa; Superdex 200 for larger).
Quality control
- SDS-PAGE at every step — shows purity progression and apparent molecular weight
- BCA or Bradford for protein concentration
- UV absorbance ratio (A260/A280) — should be near 0.6 for pure protein
- Activity assay — the only test that confirms function
- Mass spectrometry — confirms identity and purity
Common problems and fixes
- Protein in inclusion bodies: Lower induction temperature, reduce IPTG, or refold from inclusion bodies
- Protein doesn’t bind affinity column: Tag may be hidden by folding; relocate the tag
- Protein elutes early on SEC: Likely aggregated. Optimize buffer (salt, pH, additives)
- Low yield: Test expression conditions, check for proteolysis
- Contaminating bands persist: Add ion exchange or HIC step before SEC
Buffer considerations
- pH: Far enough from the protein’s pI for solubility
- Salt: 100–500 mM NaCl prevents non-specific interactions
- Reducing agents: DTT or β-mercaptoethanol if protein has free cysteines (omit on Ni columns)
- Glycerol: 10% improves stability for storage
- Detergents: Required for membrane proteins (DDM, LMNG)
Protein purification is iterative. Start with a standard tagged-protein workflow, evaluate the SDS-PAGE at each step, and add intermediate steps only when needed.
