Apr 02, 2023
Western blot - alpha-synuclein
- 1Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, UK;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, Maryland, USA.
Protocol Citation: Michael J Hurley 2023. Western blot - alpha-synuclein. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l69pqdlqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 27, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 77696
Keywords: ASAPCRN, alpha-synuclein, western blot
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000420
Abstract
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.
Materials
RIPA buffer (50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulphate)
Protease inhibitors (0.8 μM Aprotinin, 40 μM Bestatin, 140 μM E-64 at, Leupeptin at 20 μM and Pepstatin A at 15 μM, 1mM phenylmethanesulfonyl fluoride)
Towbin transfer buffer: 25 mM Tris, 192 mM glycine, pH 8.3, with 20% methanol (vol/vol).
To prepare 1 L of buffer:
- 800 mL distilled H2O
- 200 mL methanol
- 3.03 g Tris base
- 14.4 g Glycine
Sample preparation
Sample preparation
1d 4h 16m
1d 4h 16m
Sample preparation using either protein pellet from an extraction (step 1.1) or direct lysis (step 1.2)
Dissolve protein pellets from the TRI Reagent® extraction in 2% SDS containing 8 M urea.
Vortex 00:00:30 twice, then orbital shaker 260 rpm 00:30:00
30m 30s
Dissolve cells in RIPA buffer containing protease inhibitors (see Materials) On ice 00:30:00
30m
Mix samples with 4x NuPAGE™ LDS sample buffer containing 50 mM dithiothreitol
Vortex 00:00:20 twice, then orbital shaker 260 rpm 00:15:00
15m 20s
Heat samples 70 °C 00:10:00
10m
Centrifuge samples 00:00:30 to collect condensation from tube lid. Vortex 00:00:20 then centrifuge again 00:01:00
1m 50s
Electrophoresis and transfer
Electrophoresis and transfer
1d 4h 16m
1d 4h 16m
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 00:45:00 or until dye front reaches the bottom of the gel. Run with pre-stained or biotinylated size markers
45m
Wet PVDF membrane with methanol and then equilibrate in Towbin buffer (see materials)
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
Place cassette in transfer tank and transfer protein onto 0.2 mm PVDF (polyvinylidene difluoride) membranes (30 V, 01:00:00 in Towbin buffer
1h
Rinse membranes with PBS 00:00:30
30s
Immunodectection of protein bands
Immunodectection of protein bands
1d 1h 5m 30s
1d 1h 5m 30s
Fix proteins to membrane for 00:20:00 with 4% paraformaldehyde
20m
Wash with PBST (PBS containing 0.01% (v/v) Tween™-20) 4 x 00:05:00
5m
Block non-specific binding sites with block solution (PBST containing 2% (w/v) BSA and 0.005 % (w/v) thiomersal) for 01:00:00
1h
Incubate membranes in block solution containing rabbit monoclonal antibody against α-synuclein (1:1250) (ab212184) and β-actin (1:5000) (ab241153) 20:00:00 Room temperature
20h
Wash with PBST 4 x 00:05:00
5m
Incubate membrane with secondary antibody in block solution (step 15.1 or step 15.2)
Incubate membrane with biotinylated goat anti-rabbit IgG (1:1000) (Stratech) 01:15:00 and go to step 16
1h 15m
OR incubate membrane with goat anti-rabbit conjugated to peroxidase (1:25,000) (Stratech) 01:15:00 and go to step 18
1h 15m
Wash with PBST 4 x 00:05:00
5m
incubated with peroxidase conjugated streptavidin (1:1000) (Roche) 00:45:00
45m
Wash with PBST 4 x 00:05:00
5m
Wash with PBS 2 x 00:05:00
5m
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)
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 ≤ 00:00:30
30s
Rinse in running tap water > 00:05:00
5m
Dry membrane 60ºC 1 hour
For chemiluminescent detection use an Amersham ECL plus kit according to the manufacturer's instructions
Visualize ECL or digitize DAB stained membrane using a ChemiDoc™ MP imaging system (Biorad)
ECL reagents can be washed off the membrane and the membrane then processed for DAB staining if desired