Preparation of ATP13A2 microsomes from Sf9 cells

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 4°C

Wash pellet twice with 15 mL of Phosphate-buffered Saline, centrifuge at 1000x g,4°C in between washes

Gently resuspend by inverting tube and pipetting

Collect cells after final wash by centrifugation at 1500x g,4°C

Resuspend cells in 10 mL Lysis Buffer

Swell cells in Lysis Buffer by incubating on ice for 0h 10m 0s to 0h 15m 0s

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 1000x g,4°C to remove nuclear fraction and unbroken cells and save supernatant

Spin down at 10000x g,4°C (Sorvall SS-34 rotor) to remove mitochondrial-lysosomal fraction and save supernatant

Transfer supernatant to ultracentrifuge tubes and spin down at 200000x g,4°C (Beckman Type 45 Ti rotor) to collect microsomes

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