TITLE: Western blot - alpha-synuclein
AUTHORS: Michael J Hurley

[DESCRIPTION]
This protocol describes how to detect alpha-synuclein in protein derived from STC-1 cells by western blot using DAB/peroxidase or ECL to visualise the bands.

[STEPS]
SECTION: Sample preparation
1. Sample preparation using either protein pellet from an extraction (step 1.1) or direct lysis (step 1.2)
SECTION: Electrophoresis and transfer
5. Separate samples (~ 10 ug total protein)  by polyacrylamide gel electrophoresis using precast Bolt™ or NuPAGE™ 12%, Bis-Tris, 1.0 mm, Mini Protein Gels at 180 V 45 min or until dye front reaches the bottom of the gel. Run with pre-stained or biotinylated size markers

6. Wet PVDF membrane with methanol and then equilibrate in Towbin buffer (see materials)
SECTION: Sample preparation
2. Mix samples with 4x NuPAGE™ LDS sample buffer containing 50 mM dithiothreitol
Vortex 20 s twice, then orbital shaker   15 min

SECTION: Sample preparation
1.1. Dissolve protein pellets from the TRI Reagent® extraction in 2% SDS containing 8 M urea. 
Vortex 30 s twice, then orbital shaker   30 min

SECTION: Sample preparation
1.2. Dissolve cells in RIPA buffer containing protease inhibitors (see Materials) on ice 30 min

SECTION: Immunodectection of protein bands
10. Fix proteins to membrane for 20 minwith 4% paraformaldehyde

SECTION: Immunodectection of protein bands
11. Wash with PBST (PBS containing 0.01% (v/v) Tween™-20) 4 x 5 min

SECTION: Immunodectection of protein bands
12. Block non-specific binding sites with block solution (PBST containing 2% (w/v) BSA and 0.005 % (w/v) thiomersal) for 60 min

SECTION: Immunodectection of protein bands
13. Incubate membranes in block solution containing rabbit monoclonal antibody against α-synuclein (1:1250) (ab212184) and β-actin (1:5000) (ab241153) 1200 min Room temperature 

SECTION: Immunodectection of protein bands
14. Wash with PBST 4 x 5 min
SECTION: Immunodectection of protein bands
15. Incubate membrane with secondary antibody in block solution (step 15.1 or step 15.2)
SECTION: Immunodectection of protein bands
16. Wash with PBST 4 x 5 min
SECTION: Immunodectection of protein bands
17. incubated  with peroxidase conjugated streptavidin (1:1000) (Roche) 45 min

SECTION: Immunodectection of protein bands
18. Wash with PBST 4 x 5 min

SECTION: Immunodectection of protein bands
19. Wash with PBS 2 x 5 min
SECTION: Immunodectection of protein bands
20. Visualise bands with DAB if biotinylated secondary and streptavidin-peroxidase were used (step 20.1) or  use ECL plus (Amersham) if anti-rabbit peroxidase secondary was used (step 20.4)
SECTION: Immunodectection of protein bands
21. Visualize ECL or digitize DAB stained membrane using a ChemiDoc™ MP imaging system (Biorad) 
SECTION: Immunodectection of protein bands
20.1. For DAB detection incubate in PBS containing 0.05 % (w/v) 3,3′-diaminobenzidine, 0.015 % (v/v) H2O2 and 0.05 % (w/v) nickel ammonium sulphate for ≤ 30 s 

SECTION: Immunodectection of protein bands
20.3. Dry membrane 60ºC 1 hour 
SECTION: Immunodectection of protein bands
15.1. Incubate membrane with biotinylated goat anti-rabbit IgG (1:1000) (Stratech) 75 min and go to step 16

SECTION: Immunodectection of protein bands
15.2.  OR incubate membrane with goat anti-rabbit conjugated to peroxidase (1:25,000) (Stratech)  75 min and go to step 18

SECTION: Immunodectection of protein bands
20.2. Rinse in running tap water > 5 min

SECTION: Immunodectection of protein bands
22. ECL reagents can be washed off the membrane and the membrane then processed for DAB staining if desired
SECTION: Immunodectection of protein bands
20.4. For chemiluminescent detection use an Amersham ECL plus kit according to the manufacturer's instructions
8. Place cassette in transfer tank and transfer protein onto 0.2 mm PVDF (polyvinylidene difluoride) membranes (30 V, 60 min in Towbin buffer
SECTION: Sample preparation
3.  Heat samples 70 °C 10 min 
7. Soak transfer sandwich components (4 sheets of filter paper and 5 blotting pads) in Towbin transfer buffer and assemble in the transfer cassette in the following order:
Starting with the cathode plate
2 x blotting pads
2 x filter paper
gel
PVDF
2 x filter paper
3 x pad (use 3 because there is less chance of the gel slipping)
Use a roller to remove any air bubbles  

SECTION: Sample preparation
4. Centrifuge samples 30 s to collect condensation from tube lid. Vortex 20 s then centrifuge again 1 min 

9. Rinse membranes with PBS 30 s