Overview
In this video, we demonstrate a fluorescence-detection size-exclusion chromatography technique to screen for green fluorescent protein (GFP)-fused membrane protein stability in Escherichia coli following detergent solubilization. Proteins exhibiting a symmetrical peak in the size-exclusion profile with little or no free GFP indicate purified proteins with minimum degradation and aggregation, which can subsequently be selected for structure-function analysis.
Protocol
1.Solubilization and Analysis of the Membrane Fraction
- Thaw membrane fraction (if necessary), and for each detergent to be used for solubilization, aliquot 900 μl of the membrane suspension into a 1.5 ml polyallomer micro-centrifuge tube.
- Add 100 μl of each freshly prepared detergent (10% w/v solutions of DDM, DM, Cymal-6 and LDAO) to a final concentration of 1% to the specified 1.5 ml tube. For solubilization of eukaryotic membrane proteins cholesterol hemisuccinate (0.2% final concentration) can be added to the detergents.
- Incubate the mixtures at 4 °C for 1 hr with mild agitation.
- Pellet the detergent insoluble fraction with a bench top ultra-centrifuge, ensuring tubes are at least half-full, at 150,000 g at 4 °C for 45 min and retain supernatant.
NOTE: The effectiveness of detergent solubilization can be estimated using a fluorescent plate reader by measuring the GFP fluorescence of the solubilized membranes with the detergent insoluble fraction resuspended in the same volume of PBS. - Analyze the mono-dispersity of the different detergent: membrane protein complexes using fluorescence-detected size exclusion chromatography (FSEC).
- For FSEC analysis, equilibrate a size exclusion column with a broad fractionation range, e.g., 5,000 to 5,000,000 in molecular weight, with running buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.03% DDM) at a flow rate of 0.3 ml/min. Use this buffer for all samples, i.e., it is not changed for each detergent tested.
- Inject 100 μl of solubilized membranes onto the column at a flow rate of 0.3 ml/min. Monitor elution profile using fluorescence optics (excitation at 488 nm and emission at 512 nm). If an inline fluorescence monitor is not available, collect the fractions and read the fluorescence in a plate reader.
- Import elution volume and fluorescence intensity data into a spreadsheet program for graphical display.
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Materials
| Name | Company | Catalog Number | Comments |
| Cholesterol hemisuccinate | Sigma-Aldrich | C6512 | |
| CYMAL-6 (6-Cyclohexyl-1-Hexyl-βD-Maltoside) | Anatrace | C326 | |
| DDM (n-Dodecyl β-maltoside) | Generon | D97002 | |
| DM (n-Decyl β-maltoside) | Generon | D99003 | |
| LDAO (N,N-Dimethyl-ndodecylamine N-oxide) | Generon | D71111 | |
| BenchMark Fluorescent Protein Standard | Life Technologies | LC5928 | |
| Optima L-100 Ultracentrifuge running 45-Ti rotor | Beckman Coulter | 393253 | |
| Optima Max-XP ultracentrifuge running TLA-55 rotor | Beckman Coulter | 393315 | |
| Prominence UFLC with RF-20A Fluorescence detector | Shimadzu | ||
| Sepharose 6 10/300 GL size exclusion column | GE Healthcare | 17-5172-01 |
