Fluorescence size exclusion chromatography (FSEC) from ATP13A2 microsomes
Sue Sim, eunyong_park
Published: 2022-07-20 DOI: 10.17504/protocols.io.81wgb6bpolpk/v1
Abstract
Using fluorescence size exclusion chromatography (FSEC) to analyze ATP13A2 expression in microsomes
Steps
1.
Thaw 100 μg microsomes on ice
2.
Resuspend microsomes in Lysis Buffer and final volume 1% DDM/0.2% CHS (1X pellet, 3X Lysis Buffer, 1X 5% DDM/1% CHS) at 4°C
2.1.
DDM: n-dodecyl-β-D-maltopyranoside (Anatrace)
2.2.
CHS: cholesteryl hemisuccinate (Anatrace)
3.
Solubilize by rotating end-over-end for 2 h at 4C
4.
Clarify lysate by spinning at 17000x g,4°C
5.
Equilibrate Superose 6 column, connected to an HPLC system, with Running Buffer
6.
Inject 85 uL of lysate into HPLC
7.
Monitor elution of GFP-tagged ATP13A2 constructs using a fluorometer (λex= 475 nm; λem= 510 nm; with a fixed gain) connected to the system
