TITLE: Sequential extraction and immunoblotting
AUTHORS: Isabel Lam

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div><br/></div>

[STEPS]
?. [Sequential extraction and immunoblotting]
Rinse the neurons twice with PBS.
?. [Sequential extraction and immunoblotting]
Place the dish . 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:  per well for a six-well plate, and  for a 6-cm dish.
on ice
250 µl
500 µl
?. [Sequential extraction and immunoblotting]
Use a cell scraper to thoroughly scrape all neurons from each well.
?. [Sequential extraction and immunoblotting]
Place the neurons in a polyallomar tube for a table top ultracentrifuge; keep the tube .
on ice
?. [Sequential extraction and immunoblotting]
Sonicate the tube ten times at a 0.5-s pulse and at 10% power.
?. [Sequential extraction and immunoblotting]
Incubate the tube  for .
on ice
?. [Sequential extraction and immunoblotting]
Centrifuge the tube at  at  for .
Centrifuge: 100000 34
4 °C
?. [Sequential extraction and immunoblotting]
Add 4× Laemmli buffer to ~– of TX-100 supernatant . Save ~ of supernatant for protein assay. Retain the supernatant  or in a  freezer.
150 µl
200 µl
20 µl
on ice
-20 °C
Some of the supernatant is usually lost during Step 3–8; we recommend measuring the remaining supernatant before determining how much to remove for Laemmli buffer.
?. [Sequential extraction and immunoblotting]
To the pellet, add the same volume of ice-cold 1% (vol/vol) TX-100/TBS with protease and phosphatase inhibitors: per well for a six-well plate, and  for a  dish.
250 µl
500 µl
6
?. [Sequential extraction and immunoblotting]
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.
a. Sonicator: Fisherbrand™ Model 120 Sonic Dismembrator (120V, 50/60Hz)b. I used Misonix Sonicator S-4000 with Program Settings: Amplitude 10, Process Time  , Pulse-ON time:  , Pulse-OFF time:
?. [Sequential extraction and immunoblotting]
Centrifuge the mixture at  at  for .
Centrifuge: 100000 34
4 °C
?. [Sequential extraction and immunoblotting]
Discard the supernatant.
?. [Sequential extraction and immunoblotting]
Add 2% (vol/vol) SDS/TBS to the pellet with protease and phosphatases inhibitors. To a six-well plate, add  of 2% (wt/vol) SDS/TBS per well, and to a  dish add  of 2% (wt/vol) SDS/TBS.
125 µl
6
250 µl
?. [Sequential extraction and immunoblotting]
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.
a. I used Misonix Sonicator S-4000 with Program Settings: Amplitude 10, Process Time 00:00:15, Pulse-ON time: 00:00:01, Pulse-OFF time: 00:00:01
?. [Sequential extraction and immunoblotting]
Remove the supernatant and place it into a new microcentrifuge tube.
?. [Sequential extraction and immunoblotting]
Perform a BCA/protein assay on TX-100 supernatant and SDS extract. Typically, a 1:5 dilution for the BCA assay is sufficient.
?. [Sequential extraction and immunoblotting]
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).
For example, if you have   of TX-100 extract (from Step 8) at   and   of SDS extract, add   of 4× Laemmli buffer to the TX-100 extract and   of 4× Laemmli buffer to the SDS extract. Load   of both the TX-100 extract ( ) and SDS extract. We suggest using a 2× volume because it makes the insoluble a-syn species more abundant and thus easier to visualize and quantify by immunoblotting.
?. [Sequential extraction and immunoblotting]
Load the samples on a 4–20% (wt/vol) gel and run them according to the manufacturer’s directions.
We use 85 V constant voltage until the dye front runs off the gel. Be sure not to let the 10-kDa marker to run off the gel.
?. [Sequential extraction and immunoblotting]
Transfer the proteins from the gel to a nitrocellulose membrane according to the manufacturer’s instructions at 100 V for  or at 40 V.
?. [Sequential extraction and immunoblotting]
Block the membrane for  with TBS/5% (wt/vol) milk.
?. [Sequential extraction and immunoblotting]
Dilute the primary antibodies in TBS/5% (wt/vol) milk and incubate them  at  with shaking.
4 °C
?. [Sequential extraction and immunoblotting]
Rinse the membrane three times with TBS/T,  each rinse.
?. [Sequential extraction and immunoblotting]
Incubate the membrane with HRP-conjugated secondary antibodies for  at .
4 Room temperature
?. [Sequential extraction and immunoblotting]
Rinse the membrane three times with TBS/T,  each rinse.
?. [Sequential extraction and immunoblotting]
Develop with enhanced chemiluminescence.
?. [Sequential extraction and immunoblotting]
Use Misonix Sonicator S-4000, with Program Settings: Amplitude 10, Process Time , Pulse-ON time , Pulse-OFF time.
?. [Sequential extraction and immunoblotting]
Wipe sonication tip with 1% SDS after coming into contact with PFFs, then 70% ethanol.
?. [Sequential extraction and immunoblotting]
Optima MAX-TL Ultracentrifuge TLA-120.2 S/N 12U1811 .
Centrifuge: 100000 34
4 °C