May 17, 2024

Public workspaceWestern blot - alpha-synuclein

 Forked from Western blot - alpha-synuclein
DOI
dx.doi.org/10.17504/protocols.io.rm7vzjb22lx1/v1
  • 1German Center for Neurodegenerative Diseases (DZNE)
Pietro La Vitola
Open access
DOI: dx.doi.org/10.17504/protocols.io.rm7vzjb22lx1/v1
Protocol Citation: Pietro La Vitola, Eva M. Szegö 2024. Western blot - alpha-synuclein. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzjb22lx1/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 17, 2024
Protocol Integer ID: 99998
Keywords: alpha-synuclein, western blot, dosal medulla oblungata
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000420
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Abstract
This protocol describes how to detect alpha-synuclein protein in mouse dorsal medulla oblongata tissue by western blot
Materials

Laemmli SDS sample buffer, not reducing
ReagentLaemmli SDS-Sample buffer, not reducingThermo ScientificCatalog #J60660.AC


Towbin transfer buffer
25 mM Tris
192 mM glycine
Ph8.3
20% methanol (vol/vol).

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
Tissue homogenization
Homogenise samples in 150 µL ice-cold lysis buffer (1% Triton X-100 in 0.1 M phosphate buffered saline solution, Ph7.6 , supplemented with protease and phosphatase inhibitors.

Centrifuge samplesShaker14000 x g, 4°C, 00:30:00

30m
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 Temperature95 °C Duration00:05:00
5m
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 Duration01:20:00 or until dye front reaches the bottom of the gel. Run with pre-stained size markers
1h 20m
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, Duration01:30:00 in cold Towbin buffer.
1h 30m
Rinse membranes with TBS Duration00:00:30
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 Duration01:00:00
1h
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) Duration18:00:00 TemperatureRoom temperature
18h
Wash with TBS-T Duration00:05:00 4x
5m
Incubate membrane with peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG in TBS-T (1:5000 each). Duration00:00:00 RT

Wash with TBS-T Duration00:05:00 4x
5m
Incubate membrane with ECL Duration00:00:30
30s
Visualize ECL stained membrane using a BioRad ChemiDoc™.
Protocol references

CITATION
Szegö EM, Van den Haute C, Höfs L, Baekelandt V, Van der Perren A, Falkenburger BH (2022). Rab7 reduces α-synuclein toxicity in rats and primary neurons..
LINK
https://doi.org/10.1016/j.expneurol.2021.113900

Citations
Szegö EM, Van den Haute C, Höfs L, Baekelandt V, Van der Perren A, Falkenburger BH. Rab7 reduces α-synuclein toxicity in rats and primary neurons.
https://doi.org/10.1016/j.expneurol.2021.113900