Jul 20, 2022

Public workspacePreparation of ATP13A2 microsomes from Sf9 cells

DOI
dx.doi.org/10.17504/protocols.io.kxygxzde4v8j/v1
  • 1University of California, Berkeley
Sue Sim
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DOI: dx.doi.org/10.17504/protocols.io.kxygxzde4v8j/v1
Protocol Citation: Sue Sim, eunyong_park 2022. Preparation of ATP13A2 microsomes from Sf9 cells. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxzde4v8j/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: 66880
Abstract
Isolate microsomes from Sf9 cells expressing ATP13A2
Materials
Phosphate-buffered Saline (ph 7.4)
137 mM NaCl
2.7 mM KCl
10 mM Na2HPO4
1.8 mM KH2PO4

Lysis Buffer
10 mM Tris pH 7.5
0.5 mM MgCl2
2 mM DTT
0.1 mM PMSF
Plus protease inhibitors (5 µg/mL aprotinin, 5 µg/mL leupeptin, 1 µg/mL pepstatin A)

Resuspension Buffer
10 mM Tris pH 7.5
0.5 M sucrose
2 mM DTT
0.5 mM PMSF

Storage Buffer
0.25 M sucrose
Plus protease inhibitors (5 µg/mL aprotinin, 5 µg/mL leupeptin, 1 µg/mL pepstatin A, 2 mM PMSF)
Thaw Sf9 cell pellets at room temperature (typical size around 5g for 0.4 L of culture)
All subsequent steps should be carried out at Temperature4 °C

Wash pellet twice with 15 mL of Phosphate-buffered Saline, centrifuge at Centrifigation1000 x g, 4°C, 00:07:00 in between washes

7m
Gently resuspend by inverting tube and pipetting
Collect cells after final wash by centrifugation at Centrifigation1500 x g, 4°C, 00:07:00

7m
Resuspend cells in 10 mL Lysis Buffer
Swell cells in Lysis Buffer by incubating on ice for Duration00:10:00 to Duration00:15:00

25m
Lyse with Dounce homogenizer, 40 strokes tight
Dilute homogenate in equal volume Resuspension Buffer and mix
Further lyse with Dounce homogenizer, 20 strokes tight
Spin down at Centrifigation1000 x g, 4°C, 00:10:00 to remove nuclear fraction and unbroken cells and save supernatant

10m
Spin down at Centrifigation10000 x g, 4°C, 00:20:00 (Sorvall SS-34 rotor) to remove mitochondrial-lysosomal fraction and save supernatant

20m
Transfer supernatant to ultracentrifuge tubes and spin down at Centrifigation200000 x g, 4°C, 00:35:00 (Beckman Type 45 Ti rotor) to collect microsomes

35m
Resuspend microsomal pellet in Storage Buffer
Measure microsome concentrations based on total protein concentration using the Bradford Assay and bovine serum albumin as a standard
Flash-freeze in aliquots of 25-100 μL at concentrations between 2-5 mg/mL using liquid nitrogen and store at -80C until use