Jul 20, 2022
Fluorescence size exclusion chromatography (FSEC) from ATP13A2 microsomes
- 1University of California, Berkeley
Protocol Citation: Sue Sim, eunyong_park 2022. Fluorescence size exclusion chromatography (FSEC) from ATP13A2 microsomes. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgb6bpolpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: July 17, 2022
Last Modified: July 20, 2022
Protocol Integer ID: 66881
Abstract
Using fluorescence size exclusion chromatography (FSEC) to analyze ATP13A2 expression in microsomes
Materials
Lysis Buffer
50 mM Tris pH 7.5
200 mM NaCl
1 mM EDTA
1 mM DTT
10% glycerol
Plus protease inhibitors (5 µg/mL aprotinin, 5 µg/mL leupeptin, 1 µg/mL pepstatin A, and 2 mM PMSF)
Running Buffer
25 mM Tris pH 7.5
100 mM NaCl
1 mM EDTA
0.03% DDM/ 0.006% CHS
Thaw 100 μg microsomes on ice
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
DDM: n-dodecyl-β-D-maltopyranoside (Anatrace)
CHS: cholesteryl hemisuccinate (Anatrace)
Solubilize by rotating end-over-end for 2 h at 4C
Clarify lysate by spinning at 17000 x g, 4°C, 01:00:00
1h
Equilibrate Superose 6 column, connected to an HPLC system, with Running Buffer
Inject 85 uL of lysate into HPLC
Monitor elution of GFP-tagged ATP13A2 constructs using a fluorometer (λex= 475 nm; λem= 510 nm; with a fixed gain) connected to the system