Introduction to Alpha-synuclein

Protein function

• Alpha-synuclein is a neuronal protein that plays various roles in synaptic activity
• Alpha-synuclein acts as a monomer to enhance vesicle initiation, fusion, and expansion by enhancing exocytotic fusion pores, regulating synaptic vesicle transport and subsequent neurotransmitter release.
• Alpha-synuclein acts as a molecular chaperone in a multimeric membrane-bound state, assisting in the folding of the synaptic fusion component called SNARE (soluble NSF attachment protein receptor) on the presynaptic membrane, along with cysteine string protein-α/DNAJC5
• Alpha-synuclein regulates the activity of dopamine transporter protein (DAT1) by binding to it, playing a role in the regulation of dopamine neurotransmission, and is also a major component of pathogenic Lewy bodies and Lewy neurites. Mutations in its gene are associated with Parkinson's disease

Protein expression

• Alpha-synuclein is primarily expressed in the brain, with high expression at presynaptic terminals in the central nervous system
• It is also expressed in small amounts in other tissues, except the liver

Protein localization

• Alpha-synuclein is expressed in the cytoplasm, cell membrane and nucleus
• Alpha-synuclein is also secreted at cell junctions, synapses, and at the membrane binding sites of dopaminergic neurons

Figure 2: Alpha-synuclein ICC experimental result image, using the Anti-Alpha-synuclein antibody [MJFR1] (ab138501). Green: Alpha-synuclein, Red: Tubulin, Blue: DAPI.

Isoforms & post-translational modifications

• Human (P37840): Isoforms 1-3: 11-14 kDa (predicted)
• Mouse (O55042): Isoforms 1-2: 12-14 kDa (predicted)
• Rat (P37377): Isoforms 1-2: 15-16 kDa (predicted) Isoform 3: 4 kDa (predicted)
• Alpha-synuclein undergoes acetylation, phosphorylation, and ubiquitination modifications.

WB experiment tips

Precautions

• The predicted molecular weight of Alpha-synuclein is around 14kDa, but due to the presence of different isoforms and post-translational modifications, the observed band size may not be the same as the predicted value.
• Phosphorylation modification usually occurs at serine 129 and tyrosine 125 of Alpha-synuclein, so please choose the corresponding antibodies (Alpha-synuclein, Alpha-synuclein (phospho S129), Alpha-synuclein (phospho Y125)) to correctly detect unmodified or modified Alpha-synuclein.
• For the detection of phosphorylated alpha-synuclein, treating the membrane with 0.4% PFA at room temperature for 30 minutes, followed by blocking with 5% milk, helps to obtain ideal experimental results.

Positive control

• HAP1, HEK293-T cell lysate
• Human, rat brain tissue lysate
• Recombinant human Alpha-synuclein protein (ab51189)

Negative control

• HEK-293T whole cell lysate with SNCA (Alpha-synuclein) knockout (ab255433)
• HAP1 whole cell lysate with SNCA (Alpha-synuclein) knockout

Example of results

Figure 3: WB-Anti-Alpha-synuclein antibody [MJFR1] (ab138501).

Primary antibody: used ab138501 at a concentration of 1/10000.

Lane 1: Wild-type HAP1 whole cell lysate.
Lane 2: SNCA knockout HAP1 whole cell lysate.
Lane 3: Human brain tissue lysate.
Lane 4: Mouse brain tissue lysate.

Predicted band size: 14 kDa.

Key control points

In the experiment, in addition to paying attention to routine issues, it is also essential to pay special attention to the following key control points:

Sample preparation:

1. Add a complex protease inhibitor to avoid degradation of the target protein.
2. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Transferring:

1. We strongly recommend using Coomassie Brilliant Blue staining after transferring to confirm the success of the transfer.

Antibody incubation:

1. We recommend using fresh antibodies and avoid reusing them.

Reference

1. Todd Logan, Jacob Bendor, Chantal Toupin, Kurt Thorn, Robert H Edwards. α-Synuclein promotes dilation of the exocytotic fusion pore. Nat Neurosci. 2017 May;20(5):681-689. doi: 10.1038/nn.4529. Epub 2017 Mar 13.
2. Jacqueline Burré, Manu Sharma, Theodoros Tsetsenis, Vladimir Buchman, Mark R Etherton, Thomas C Südhof. Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro. Science. 2010 Sep 24;329(5999):1663-7. doi: 10.1126/science.1195227. Epub 2010 Aug 26.
3. Brittany Butler, Kaustuv Saha, Tanu Rana, Jonas P Becker, Danielle Sambo, Paran Davari, J Shawn Goodwin, Habibeh Khoshbouei. Dopamine Transporter Activity Is Modulated by α-Synuclein. J Biol Chem. 2015 Dec 4;290(49):29542-54. doi: 10.1074/jbc.M115.691592. Epub 2015 Oct 6.
4. M S Goldberg, P T Lansbury Jr. Is there a cause-and-effect relationship between alpha-synuclein fibrillization and Parkinson's disease? Nat Cell Biol. 2000 Jul;2(7):E115-9. doi: 10.1038/35017124.