Protocol to Purify Aggregates from Patients Brain

Sarkosyl Extraction of aSyn Fibrils from Human Brain Tissue for Structural Studies

Make 10% (w/v) brain homogenate in buffer I using Precellys tubes with bead homogenizer. *Program: 6,000 rpm, 30 sec. Repeat 3 times. Keep tubes on ice.

  1. Centrifuge the Precellys tubes at 10,000 x g for 10 min at 4 °C and transfer the supernatant into another tube (on ice).
  2. Add the same amount of buffer I to the debris in the Precellys tube and re-homogenize it.
  3. After 2nd homogenization, remove the total homogenate and combine it with the supernatant from step 2. Pipette to mix.
  4. Add sarkosyl to the combined sample from step 4 to reach the final concentration of 2% sarkosyl.
  5. Incubate at 37°C for 30 min with agitation at 200 rpm.
  6. Centrifuge the homogenate-sarkosyl mixture at 10,000 x g for 10 min at room temp.
  7. Transfer the supernatant to another centrifuge tube and centrifuge one more time at 10,000 x g for 10 min at room temp (RT).
  8. Transfer the supernatant to an ultracentrifuge tube and centrifuge at 100,000 x g for 60 min at 4 o Discard the supernatant and resuspend the pellet in 500 uL per gram of tissue (i.e. if the tissue weight is around 0.2 gram, then dissolve the pellet in 100 uL) in Buffer II and keep it at 4 °C.
  9. Centrifuge the resuspended pellet at 2,000 x g for 20 min at RT (if buffer contains sucrose, then centrifuge at 10,000 x g).
  10. Centrifuge the supernatant at 100,000 x g for 1 hour at 4 o
  11. Resuspend the pellet in 25 uL per gram of tissue with Buffer III and store it at -80 o
  12. On day of imaging, centrifuge the sample at 1,000 x g for 1 min and prepare grid with the supernatant.

Buffer I: 10 mM Tris-HCl (pH 7.5), 0.8 M NaCl, 10% sucrose, 1 mM EGTA, 1x protease inhibitors, 1x phosphatase inhibitors (Beta mercaptoethanol (BME) will be added right before the homogenization to reach the final concentration of 40 mM).

Buffer II: Buffer I (without sucrose) + sarkosyl

Buffer III: 20 mM Tris (pH 7.5), 100 mM NaCl