Fluorescence-Detection Size-Exclusion Chromatography: A Technique to Identify the Integrity of Fluorescent Membrane Proteins upon Detergent Solubilization

Samenvatting

Source: Bird, L. E., et al. Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli. J. Vis. Exp. (2015)

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

1. 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.
2. 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.
3. Incubate the mixtures at 4 °C for 1 hr with mild agitation.
4. Pellet the detergent insolub....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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