Endosome isolation

prepare 0.5M and 2M sucrose buffer

prepare buffer S2 by mixing 0.5M and 2M sucrose at 1:2 and buffer S3 at 2:1 ratio

place Beckman ultracentrifuge tube (No.344059) on dry ice

add 2.5ml 2M sucrose in the tube and wait for 0h 10m 0s or until it frozen.

add 2.5 ml buffer S2 on top of frozen 2M sucrose and wait for 0h 10m 0s or until it frozen

add 2.5 ml buffer S3 on top of frozen buffer S2 and wait for 0h 10m 0s or until it frozen

add 2.5 ml 0.45M sucrose on top of frozen buffer S3 and wait for 0h 10m 0s or until it frozen

keep the frozen tubes at -80 until experiment day

place the frozen tubes at 4C for 5h 0m 0s or until defrosted before use

Dissect striatum from mice, fresh freeze the tissue with dry ice

Prepare homogenization buffer (HB): 150mM Nacl, 10mM HEPES, 1mM EGTA, 0.1M MgCl

Homogenize striatum tissue with 1ml HB containing 1x Halt protease and phosphatase inhibitor (EDTA-free) by Dounce homogenization on ice (30 strokes)

Centrifugation 0h 10m 0s, 3,000g, 4C

load postnuclear supernatants onto the top of continuous sucrose gradient

Centrifugation 18h 0m 0s, 200,000g, 4C (Accel and Decel -> slow)

Collect fractions from top to bottom of the gradient

add equal volume of ice-cold 55% TCA and incubate on ice for 0h 30m 0s

centrifugation 0h 10m 0s, 20,000g, 4C

wash pellet with 1ml ice-cold acetone centrifugation 0h 10m 0s, 20,000g, 4C

for 2 times. Total 3 washes

resuspend pellet with 70ul of 1% SDS buffer (1% SDS, 10mMTris, 2mMEDTA) containing 1x Halt protease and phosphatase inhibitor (EDTA-free), sonication for 0h 0m 3s and followed by incubation on orbital shaker for 1hr at room temperature.

Store samples at -80 for western blot assay