Fluorescence-Detection Size-Exclusion Chromatography: A Technique to Identify the Integrity of Fluorescent Membrane Proteins upon Detergent Solubilization
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 concentrat…
Fluorescence-Detection Size-Exclusion Chromatography: A Technique to Identify the Integrity of Fluorescent Membrane Proteins upon Detergent Solubilization. J. Vis. Exp. (Pending Publication), e21101, doi: (2023).