Sequential extraction and immunoblotting
Isabel Lam
Abstract
This protocol examines the fraction of alpha-synuclein (as assessed by alpha-synuclein and/or PS129 western blot) that is present in the triton-soluble or SDS-soluble fraction. Addition of alpha-synuclein pre-formed fibrils (PFFs) to neuron cultures seeds the recruitment of endogenous or transgenic alpha-synuclein into aggregates characterized by detergent insolubility. Monomeric alpha-synuclein will be present in the triton-soluble fraction, whereas PFF-induced alpha-synuclein oligomers will be present in the triton-insoluble, SDS-soluble fraction. This protocol encompasses preparation of cell extracts with Triton X-100, followed by sequential extraction of the Triton-insoluble material with SDS. The protein in the different detergent fractions are quantified by BCA, followed by SDS-PAGE and WB for alpha-synuclein, PS129, TUJ1, and loading control such as GAPDH.
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Sequential extraction and immunoblotting
Rinse the neurons twice with PBS.
Place the dish On ice. By working one well at a time, completely aspirate the PBS and add the following volumes of ice-cold 1% (vol/vol) TX-100/TBS with protease and phosphatase inhibitors: 250µL per well for a six-well plate, and 500µL for a 6-cm dish.
Use a cell scraper to thoroughly scrape all neurons from each well.
Place the neurons in a polyallomar tube for a table top ultracentrifuge; keep the tube On ice.
Sonicate the tube ten times at a 0.5-s pulse and at 10% power.
Use Misonix Sonicator S-4000, with Program Settings: Amplitude 10, Process Time 0h 0m 10s, Pulse-ON time 0h 0m 1s, Pulse-OFF time0h 0m 1s.
Wipe sonication tip with 1% SDS after coming into contact with PFFs, then 70% ethanol.
Incubate the tube On ice for 0h 30m 0s.
Centrifuge the tube at 100000x g,0h 0m 0s at 4°C for 0h 30m 0s.
Optima MAX-TL Ultracentrifuge TLA-120.2 S/N 12U1811 100000x g,0h 0m 0s 0h 30m 0s 4°C.
Add 4× Laemmli buffer to ~150µL–200µL of TX-100 supernatant . Save ~20µL of supernatant for protein assay. Retain the supernatant 4On ice or in a -20°C freezer.
To the pellet, add the same volume of ice-cold 1% (vol/vol) TX-100/TBS with protease and phosphatase inhibitors: 250µLper well for a six-well plate, and 500µL for a 6cm dish.
Sonicate ten times at a 0.5-s pulse and at 10% power. Keep the tip of the probe toward the bottom of the tube to prevent frothing. Make sure that the pellet is completely dispersed.
Centrifuge the mixture at 100000x g,0h 0m 0s at 4°C for 0h 30m 0s.
Discard the supernatant.
Add 2% (vol/vol) SDS/TBS to the pellet with protease and phosphatases inhibitors. To a six-well plate, add 125µL of 2% (wt/vol) SDS/TBS per well, and to a 6cm dish add 250µL of 2% (wt/vol) SDS/TBS.
Sonicate 15 times, at a 0.5-s pulse and at 10% power. Keep the tip of the probe toward the bottom of the tube. Make sure that the pellet is completely dispersed.
Remove the supernatant and place it into a new microcentrifuge tube.
Perform a BCA/protein assay on TX-100 supernatant and SDS extract. Typically, a 1:5 dilution for the BCA assay is sufficient.
Dilute 2% (wt/vol) SDS extract from Step 15 into Laemmli buffer to 2× volume for the corresponding TX-100 fraction (regardless of the protein concentration of the SDS fraction).
Load the samples on a 4–20% (wt/vol) gel and run them according to the manufacturer’s directions.
Transfer the proteins from the gel to a nitrocellulose membrane according to the manufacturer’s instructions at 100 V for 1h 15m 0s or 1h 15m 0sat 40 V.
Block the membrane for 1h 0m 0s with TBS/5% (wt/vol) milk.
Dilute the primary antibodies in TBS/5% (wt/vol) milk and incubate them 1h 0m 0s at 4°C with shaking.
Rinse the membrane three times with TBS/T, 0h 10m 0s each rinse.
Incubate the membrane with HRP-conjugated secondary antibodies for 1h 0m 0s at 4Room temperature.
Rinse the membrane three times with TBS/T, 0h 10m 0s each rinse.
Develop with enhanced chemiluminescence.
