Protocol of Ionic Detergents Isolate Membrane Protein: Step-by-Step Implementation

Complete Workflow from Cells to Pure Protein

Stage A: Membrane Preparation

1. Cell harvest: Centrifuge at 5,000×g, 4°C, 10 min
2. Lysis: French press at 15,000 psi or sonication (3×30 sec bursts)
3. Membrane enrichment: 100,000×g ultracentrifugation for 1 hour
4. Wash: Resuspend pellet in 50 mM Tris pH 8.0, 150 mM NaCl, repeat centrifugation
5. Quantify: Bradford assay, target 5-10 mg/mL membrane protein

Stage B: Detergent Screening (24-well format)

1. Prepare stocks: 10× concentrated detergents in same buffer
2. Dispense: 95 μL membrane suspension + 5 μL detergent stock per well
3. Incubate: 30 min at 4°C with gentle mixing
4. Clarify: Microcentrifuge at 20,000×g for 20 min
5. Assess:
   • Soluble yield: SDS-PAGE of supernatant
   • Activity: Ligand binding or functional assay
   • Select best condition: Target >50% extraction, >60% activity retention

Stage C: Scale-Up Solubilization

1. Transfer optimal condition: Scale to 10-50 mL
2. Incubation: 30-60 min at 4°C with stirring
3. Ultracentrifuge: 100,000×g for 45 min
4. Recovery: Collect supernatant carefully, avoid lipid layer

Stage D: Affinity Purification (IMAC Example)

1. Column equilibration: 5 CV of buffer A (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% optimal detergent)
2. Sample loading: Supernatant at 0.5-1 mL/min flow rate
3. Washing: 10-15 CV buffer A to remove unbound detergent
4. Elution: Gradient to buffer B (buffer A + 250 mM imidazole)
5. Peak collection: Monitor A₂₈₀, pool fractions

Stage E: Detergent Exchange (if needed)

1. Concentration: Amicon Ultra 100 kDa MWCO to <2 mL
2. Exchange buffer: 50 mM Tris pH 8.0, 0.05% DDM (target mild detergent)
3. Repeated dilution/concentration: 3× cycles to achieve >95% exchange
4. Final concentrate: 0.5-2 mL at 1-5 mg/mL

Stage F: Polishing SEC

1. Column preparation: Superdex 200 Increase 10/300 GL, equilibrate with 2 CV SEC buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.05% DDM)
2. Injection: 0.5 mL sample
3. Elution: 0.5 mL/min flow rate, collect 0.5 mL fractions
4. Analysis: SDS-PAGE every other fraction, pool monodisperse peak

Stage G: Quality Control

1. Concentration: A₂₈₀ measurement, calculate yield
2. Purity: SDS-PAGE with Coomassie staining, target >95%
3. Monodispersity: DLS measurement, target polydispersity <15%
4. Activity: Functional assay (binding, transport, catalysis)
5. Stability: Thermal shift assay, measure Tm

Expected Timeline

• Day 1: Membrane prep, screening setup
• Day 2: Scale-up solubilization, IMAC
• Day 3: Detergent exchange, SEC
• Day 4: QC and activity assays

Total hands-on time: 12-16 hours over 4 days

Troubleshooting: Comprehensive Problem-Solving Guide

Problem	Potential Cause	Diagnostic Test	Solution
No protein in supernatant	Detergent concentration too low	Pilot with 5×CMC	Increase detergent or add chaotrope (2 M urea)
	Protein in insoluble aggregates	SDS-PAGE of pellet	Try mixed detergents or sonication
Protein doesn't bind IMAC	Detergent strips His-tag	Western blot tag integrity	Reduce detergent or add 10 mM imidazole during solubilization
	Charge interference from anionic detergent	Test binding in non-ionic	Dilute sample 10-fold or add 100 mM NaCl
Multiple SEC peaks	Aggregation	DLS of peak fractions	Add stabilizers (glycerol, arginine) or reduce concentration
	Lipid contamination	Lipid stain of SDS-PAGE	Increase wash stringency or add lipid removal step
Activity loss >50%	Over-solubilization	Time-course assay	Reduce incubation time and temperature
	Essential lipid removal	Add back lipids	Supplement solubilization buffer with 0.1% synthetic lipids
	Oxidative damage	Check for free cysteines	Add 1-5 mM TCEP or DTT throughout
CTAB precipitation	High ionic strength	Conductivity measurement	Reduce NaCl to <50 mM or switch to DTAB
	Temperature fluctuation	Visual inspection	Maintain strictly at 4°C, avoid freeze-thaw