Complex Formation Skill
Guidelines for assembling and purifying macromolecular complexes for structural biology.
Gel Filtration (Size Exclusion Chromatography)
Standard Setup
| Parameter | Value |
|---|---|
| Column | Superose 6 Increase 3.2/300 |
| System | ÄKTA micro |
| Fraction size | 0.05 mL (50 µL) |
| Void volume | B6 |
Elution Positions by Complex Size
| Complex | Expected Fractions | Notes |
|---|---|---|
| Large EC* (full elongation complex) | B4-B5 | DSIF, SPT6, PAF1c, etc. |
| EC* + nucleosome | B5-B6 | May approach void |
| Minimal EC (Pol II + DSIF) | B5-C3 | Intermediate |
| Pol II only | C4-C6 | ~500 kDa reference |
Rule: If complex elutes earlier than expected → possible aggregation. If later → incomplete assembly or dissociation.
A260/A280 Ratio Guidelines
| Complex Type | Expected Ratio | Interpretation |
|---|---|---|
| EC* on linear DNA | A280 > A260 | Protein dominates signal |
| Nucleosome-containing | A260 >> A280 | Nucleosomal DNA dominates |
Red flags:
- •A260/A280 inverted from expectation → wrong complex or contamination
- •Very high A260 → free nucleic acid contamination
Concentration Targets
| Application | Target Range | Minimum |
|---|---|---|
| Cryo-EM grids | 100-200 nM | 60 nM |
| Transcription elongation assays | 100-200 nM | 60 nM |
Below 60 nM: Not usable — concentration too low for downstream applications.
Quality Control Checkpoints
1. GF Trace Analysis
- •Check elution volume matches expected MW
- •Verify A260/A280 ratio is appropriate for complex type
- •Look for symmetric peak (asymmetry suggests heterogeneity)
2. SDS-PAGE
- •Always run SDS-PAGE on fractions before proceeding
- •Check for:
- •All expected subunits present
- •Correct stoichiometry (band intensities)
- •No unexpected bands (contamination/degradation)
- •No missing subunits (incomplete assembly)
3. Fraction Selection
- •Use single fractions (do not pool)
- •Selected fractions get cross-linked before grid preparation
Complex Assembly Workflow
- •Prepare components — Query LabBook for current stock concentrations
- •Calculate recipe — Use
complex_cli.pywith appropriate preset - •Mix components — Follow stoichiometry from preset (typically 1.5× excess over Pol II)
- •Incubate — Allow complex formation (timing depends on complex)
- •GF purification — Superose 6 Increase 3.2/300
- •Analyze fractions — Check trace + run SDS-PAGE
- •Select fraction — Based on elution position, A260/A280, gel appearance
- •Cross-link — If proceeding to cryo-EM
- •Grid preparation — At 100-200 nM
CLI Tools
bash
# List available presets python3 scripts/complex_cli.py list # Calculate recipe for EC* python3 scripts/complex_cli.py ec_star --pol2 5.2uM --volume 100 # Calculate TC-NER complex python3 scripts/complex_cli.py tc_ner --pol2 5.2uM --volume 100
Troubleshooting
| Problem | Possible Cause | Solution |
|---|---|---|
| Elutes at void (B6) | Aggregation | Reduce concentration, add salt, check buffer |
| Elutes later than expected | Incomplete assembly | Check component activity, increase incubation |
| Missing bands on gel | Component limiting | Verify stock concentrations, check stoichiometry |
| Extra bands on gel | Contamination/degradation | Check protein quality, use fresh stocks |
| Low A280 signal | Low yield | Scale up input, optimize assembly conditions |
Empirical Knowledge
Column calibration is empirical — based on previous runs with known complexes. No formal MW standards are used; positions are learned from experience.
Last updated: 2026-01-17