TITLE: pS129 alpha-synuclein Western blot
AUTHORS: Christopher Burke

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the investigation of the amount of total alpha-synuclein and pS129 alpha-synuclein in cells.</div></div>

[STEPS]
?. [DETERGENT-BASED CELL LYSIS]
Add  of lysis buffer to the cell pellet and mix thoroughly by pipetting up and down until the cell pellet is completely resolved into a homogeneous solution.
100 µl
?. [DETERGENT-BASED CELL LYSIS]
Adjust the lysis buffer volume to the cell pellet size, keep the sample at  as soon as the lysis buffer has been added, and work quickly to prevent proteolysis.
0 Room temperature
?. [DETERGENT-BASED CELL LYSIS]
Starting with a frozen cell pellet, keep it on dry ice initially and thaw it on regular ice as soon as everything is set up.
?. [DETERGENT-BASED CELL LYSIS]
If the cell pellet is collected right before conducting the Western blot, keep it  until the lysis buffer is added.
on ice
?. [DETERGENT-BASED CELL LYSIS]
Vortex and heat samples for  at .
100 °C
?. [DETERGENT-BASED CELL LYSIS]
Spin down at maximal speed for . Transfer the supernatant to a new Eppendorf tube and discard the cell pellet.
?. [DETERGENT-BASED CELL LYSIS]
Store the supernatant at  in order to proceed with the protocol at a later point or as a positive control for later experiments.
-20 °C
?. [DETERGENT-BASED CELL LYSIS]
Quantify protein concentration through a classical BCA assay (or other available techniques).
?. [WESTERN BLOT]
For each sample, mix an appropriate amount of protein with NuPAGE® LDS Sample Buffer (4x), reducing agent (10x) and sterile H2O for protein linearization and monomerization.
?. [WESTERN BLOT]
Load no more than  protein per well (e.g. ).
40 µg
30 µg
?. [WESTERN BLOT]
Heat samples for  at .
95 °C
?. [WESTERN BLOT]
Prepare  of running buffer 1X by mixing  diH20 with  NuPAGE® MES SDS Running Buffer (20x).
500 mL
475 mL
25 mL
?. [WESTERN BLOT]
Place a 4-12 % Bis-Tris Gel in the gel electrophoresis chamber (after removing the tape and the comb and after rinsing) and fill the chamber with running buffer.
?. [WESTERN BLOT]
Use gel-loading tips to load the samples and  of the protein ladder (Precision Plus Protein™ Dual Color Standards) onto the gel.
4 µl
?. [WESTERN BLOT]
Run the gel at 150 V for approximately  –  until the protein ladder.
The loading dye indicate a sufficient electrophoretic separation.
?. [WESTERN BLOT]
Use the iBlot Gel Transfer Device and the iBlot PVDF Transfer Stacks for dry blotting of proteins.
?. [WESTERN BLOT]
Boot the gel transfer device, select program “P0 7 min” (20v 1:00; 23v 4:00; 25v 2:00) and place the iBlotanode stack (“bottom”), with the PVDF transfer membrane on top, into the gel transfer device.
?. [WESTERN BLOT]
Separate the two plastic plates of the gel cassette using a gel knife. Cut off excessive parts of the gel. Transfer the gel onto the PVDF membrane. Gently remove any air bubbles using a wetted blotting roller.
?. [WESTERN BLOT]
Wet a Whatman paper in diH2O and place it on top of the gel. Gently remove any air bubbles using the wetted blotting roller.
?. [WESTERN BLOT]
Place the iBlot cathode stack (“top”) on top and even the layers with the blotting roller. Place the iBlot sponge on the inner side of the iBlot lid in the right orientation.
?. [WESTERN BLOT]
Close the iBlot gel transfer device and start the program.
?. [FIXATION, BLOCKING AND ANTIBODY INCUBATION]
Fix membrane in 4% paraformaldehyde in PBS for  rocking at .
95 Room temperature
?. [FIXATION, BLOCKING AND ANTIBODY INCUBATION]
Wash 3X with TBS (use and discard paraformaldehyde in compliance with the laboratory safety regulations).
?. [FIXATION, BLOCKING AND ANTIBODY INCUBATION]
Wash the PVDF membrane by gently rocking it in TBS for .
?. [FIXATION, BLOCKING AND ANTIBODY INCUBATION]
When transferring the PVDF membrane or exchanging the solution in the blot box, make sure that the PVDF membrane never dries out.
?. [FIXATION, BLOCKING AND ANTIBODY INCUBATION]
Remove the TBS and add a sufficient volume of one part Odyssey blocking buffer (TBS) mixed with one part of TBS (1:1). Incubate for  on a rocker.
?. [FIXATION, BLOCKING AND ANTIBODY INCUBATION]
Exchange the blocking solution with the primary antibody solution, i.e. one part Odyssey blocking buffer (TBS) mixed with one part TBS + the primary antibodies at the desired dilution.
?. [FIXATION, BLOCKING AND ANTIBODY INCUBATION]
Incubate at  on a rocker .
4 °C
?. [FIXATION, BLOCKING AND ANTIBODY INCUBATION]
Wash the PVDF membrane with TBS + 0.01% Tween20 on a rocker 3 times for .
?. [FIXATION, BLOCKING AND ANTIBODY INCUBATION]
Exchange with the secondary antibody solution, i.e. one part Odyssey blocking buffer (TBS) mixed with one part TBS (1:1) + the secondary antibody at the desired dilution.
?. [FIXATION, BLOCKING AND ANTIBODY INCUBATION]
Incubate on a rocker for  at  protected from light, i.e. wrap the blot box in aluminum foil.
4 Room temperature
?. [FIXATION, BLOCKING AND ANTIBODY INCUBATION]
Wash the PVDF membrane with TBS + 0.01% Tween20 on a rocker 3 times for  protected from light.
?. [FIXATION, BLOCKING AND ANTIBODY INCUBATION]
Wash the PVDF membrane again with TBS until Tween-20 has been entirely removed and scan the blot using the Odyssey CLx Infrared Imager; for bands quantification use the Odyssey Software version 3.0.