PK Digestion of-SAA Amplified Aggregates

  1. Prepare 10x PK master mix:

a. Prepare 10 mg/mL stock of Proteinase K (Sigma; Cat# P2308) dissolved in nanopure water. Gently invert to mix. Do not vortex.

b. Dilute 10x PK stock to 2x using PBS (10x) and nanopure water. For each sample to be digested, use the following volumes:

Stock Concentration Volume (uL) Final MM Concentration
PK 10x stock 10 mg/mL 2 2 mg/mL
PBS 10x 10x 2 2 x
Nanopure water 6
Final 10 2x

Note: Pipette mix gently. Do not vortex.

  1. Mix 10 µL of 2x PK master mix with 10 µL of PMCA amplified material in a low binding Eppendorf tube. Pipette mix gently.
  2. Incubate sample in thermomixer set to 37 oC and 200 rpm for 90 minutes.
  3. Stop PK digestion reaction by adding 20 µL of NuPage 2x sample buffer. Boil at 95oC for 10 minutes.
  4. Run SDS-PAGE (load 10 µL sample) and transfer to nitrocellulose membrane.
  5. Block membrane with 5% milk in PBS-T (0.1%) for 1 hour at room temperature.
  6. Blot with anti-αSyn (BD clone 42) diluted 1:5000 in blocking buffer.
  7. Wash membrane 3x with PBS-T (0.1%), 10 minutes each.
  8. Blot with anti-mouse (Sigma) diluted 1:10000 in blocking buffer.
  9. Wash membrane 3x with PBS-T (0.1%), 10 minutes each.
  10. Develop with ECL and image.