Western blot - alpha-synuclein
Pietro La Vitola, Eva M. Szegö
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Abstract
This protocol describes how to detect alpha-synuclein protein in mouse dorsal medulla oblongata tissue by western blot
Steps
Sample preparation
Tissue homogenization
Homogenise samples in 150 µL ice-cold lysis buffer (1% Triton X-100 in 0.1 M phosphate buffered saline solution, 7.6 , supplemented with protease and phosphatase inhibitors.
Centrifuge samples14000x g
Transfer supernatants into pre-cooled test tubes.
Measure protein concentration (e.g. BCA method).
Mix 4 µg samples in Laemmli sample buffer supplemented with 5 % beta-mercaptoethanol.
Heat samples 95°C 0h 5m 0s
Electrophoresis and transfer
Separate samples (4 µg total protein) by polyacrylamide gel electrophoresis using precast Bolt™ 4-20% Bis-Tris, 1.0 mm, Mini Protein Gels at 120 V 1h 20m 0s or until dye front reaches the bottom of the gel. Run with pre-stained size markers
Soak nitrocellulose membrane (pore size: 0.2 µm) with Towbin buffer (see materials)
Soak transfer sandwich components (2 sheets of filter paper and 2 blotting pads) in Towbin transfer buffer and assemble in the transfer cassette in the following order:
cathode plate
1 x blotting pad
1 x filter paper
gel
nitrocellulose membrane
1 x filter paper
1 x pad
Use a roller to remove any air bubbles
Place cassette in transfer tank and transfer protein onto 0.2 µm nitrocellulose membranes (300 mA, 1h 30m 0s in cold Towbin buffer.
Rinse membranes with TBS 0h 0m 30s
Immunodetection of protein bands
Block non-specific binding sites with blocking solution (TBS-T: TBS containing 2% BSA and 0.05 % Tween-20) for 1h 0m 0s
Incubate membranes in blocking solution containing mouse anti-α-synuclein (1:1000; RRID:AB_398108) and rabbit anti-β-actin (1:1000; RRID:AB_2305186) 18h 0m 0s Room temperature
Wash with TBS-T 0h 5m 0s
Incubate membrane with peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG in TBS-T (1:5000 each).
Wash with TBS-T 0h 5m 0s
Incubate membrane with ECL 0h 0m 30s
Visualize ECL stained membrane using a BioRad ChemiDoc™.
