Soluble and insoluble A-SYN fractionation
Hariam Raji, michela.deleidi, Maria Jose Perez J., Pascale Baden, Federico Bertoli
Abstract
Soluble/insoluble alpha-synuclein fractionation is a technique used to separate different forms of the alpha-synuclein protein based on their solubility properties.
Attachments
Steps
Soluble and insoluble A-SYN fractionation
Perform extraction and detection of Triton-soluble (T-sol) and Triton-insoluble (T-insol) alpha-synuclein as described in Stojkovska and Mazzulli 53.
Lyse individual organoids in 1% Triton X-100 extraction buffer supplemented with 1X PIC, 50millimolar (mM) NaF, 2millimolar (mM) NA3VO4 and 0.5millimolar (mM) PMSF.
Extraction buffer
| A | B |
|---|---|
| Triton X-100 | 1% |
| NaCl | 150 mM |
| glycerol | 10% |
| HEPES pH 7.4 | 25 mM |
| EDTA | 1 mM |
| MgCl2 | 1.5 mM |
Homogenize samples with a pestle and incubate on a platform shaker in an ice-water slurry for 0h 30m 0s, followed by three freeze/thaw cycles and ultracentrifugation at 100000x g,4°C.
Collect the supernatant (Triton-X Soluble fraction).
Wash the remaining pellet in Triton X-100 extraction buffer followed by another ultracentrifugation at 100000x g,0h 0m 0s.
Resuspend the pellet in 2% SDS buffer containing 50millimolar (mM) Tris, 7.4 and 1X PIC, boil it for 0h 10m 0s at 100°C (Triton-X insoluble Fraction) and label the T-insol fraction.
Sonicate Tx-Insoluble samples for 0h 10m 0s at 30% power, 20C in a cup horn sonicator (Qsonica-Q700), and then boil them again for 0h 10m 0s at 100°C.
Ultracentrifuge Tx-Insoluble samples at 100000x g,21°C.
Collect the supernatant (SDS-soluble fraction).
Detect protein concentrations using a BCA assay and load 30µg of total protein for each condition.
