α synuclein

Manufactured by Merck Group

Sourced in United States

α-synuclein is a protein that is commonly used in research laboratories. It plays a role in the regulation of neurotransmitter release and is associated with Parkinson's disease. This product is intended for research use only and its core function is to serve as a tool for studying the properties and behavior of this protein.

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Lab products found in correlation

Ubiquitin, by Cell Marque (2 mentions) Cd68 kp 1, by Roche (1 mentions) 3 amino 9 ethylcarbazol hrp substrate kit, by Vector Laboratories (1 mentions) Gill s hematoxylin, by Vector Laboratories (1 mentions) Aβ 4g8, by BioLegend (1 mentions) Permount mounting medium, by Thermo Fisher Scientific (1 mentions) Benchmark ultra, by Roche (1 mentions) Anti active caspase 3, by Cell Signaling Technology (1 mentions) α tubulin, by Merck Group (1 mentions) Ecl chemiluminescence system, by Thermo Fisher Scientific (1 mentions) Pvdf membrane, by Thermo Fisher Scientific (1 mentions) Gapdh, by Merck Group (1 mentions) Anti h3, by Abcam (1 mentions) Ripa buffer, by Merck Group (1 mentions) Lysis buffer, by Ozyme (1 mentions) Nigericin, by Merck Group (1 mentions) Nigericin, by InvivoGen (1 mentions) Bay11 7082, by Merck Group (1 mentions) Vx 765, by InvivoGen (1 mentions) E coli 0111 b4, by InvivoGen (1 mentions)

11 protocols using α synuclein

Fibrillation Protocols for Amyloid Peptides

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For Aβ1–42 fibrils, Aβ1–42 (1 mg) was dissolved in 200 μl 10 mM NaOH (5 mg/ml). Aliquots (100 μl) were diluted in 400 μl PBS 1.25× (final concentration 1 mg/ml) and were incubated at 37 °C for 72 h.
For islet amyloid polypeptide (IAPP) fibrils, 1 mg IAPP (Anaspec, reference 60,804) was dissolved in 200 μl dimethylsulfoxide (DMSO) 50% (5 mg/ml). Aliquots of 100 μl were diluted in 400 μl PBS 1.25×. Samples were incubated at 37 °C for 72 h.
For α-synuclein fibrils, α-synuclein (Millipore, reference AG938) was provided lyophilized in Tris-HCl buffer, pH 7.4, and 500 μg α-synuclein were dissolved in 200 μl H₂O MilliQ + 50 μl 500 mM NaCl solution. Samples were incubated by shaking at 37 °C for 7 days (750 rpm).
After the incubation, samples were centrifuged at 17,000 g for 20 min at 4 °C. The supernatants were discarded. Pellets of synuclein fibrils were washed twice with 200 μl PBS and then resuspended in 100 μl PBS, and the fibril sample concentration was determined using the Bradford test.

Pradier L., Blanchard-Brégeon V., Bohme A., Debeir T., Menager J., Benoit P., Barneoud P., Taupin V., Bertrand P., Dugay P., Cameron B., Shi Y., Naimi S., Duchesne M., Gagnaire M., Weeden T., Travaline T., Reczek D., Khiroug L., Slaoui M., Brunel P., Fukuyama H., Ravetch J., Canton T, & Cohen C. (2018). SAR228810: an antibody for protofibrillar amyloid β peptide designed to reduce the risk of amyloid-related imaging abnormalities (ARIA). Alzheimer's Research & Therapy, 10, 117.

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Post-mortem Neuropathological Examination

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The autopsy was carried out five hours after death. The right hemisphere was cut into coronal sections, frozen on dry ice, and stored at − 80 °C until use, while the left hemisphere was fixed in 4% buffered formalin for 15 days and used for microscopic examination.
After fixation, coronal sections of the left hemisphere were treated with formic acid and then post-fixed in formalin and embedded in paraffin. Then, cut at 5 µm and stained with hematoxylin and eosin, Congo Red and Thioflavin S. Immunohistochemistry was carried out using the following primary antibodies against: PrP (3F4, Dako) (with pretreatment with PK), β-amyloid 1–40 and β-amyloid 1–42 (from Dr M. Sarasa, Zaragoza, Spain), hyperphosphorylated tau (AT8; Innogenetics), 3R tau (RD3, clone 8E6/C11, Merck) (1:3000) and 4R tau (RD4, clone 1E1/A6, Merck) (1:100), glial fibrillary acidic protein (GFAP) (EP672Y, Roche), CD68 (KP-1, Roche) and α-synuclein (Chemicon).

Eraña H., San Millán B., Díaz-Domínguez C.M., Charco J.M., Rodríguez R., Viéitez I., Pereda A., Yañez R., Geijo M., Navarro C., Perez de Nanclares G., Teijeira S, & Castilla J. (2022). Description of the first Spanish case of Gerstmann–Sträussler–Scheinker disease with A117V variant: clinical, histopathological and biochemical characterization. Journal of Neurology, 269(8), 4253-4263.

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Immunohistochemical Analysis of Neurodegenerative Markers

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Immunohistochemistry was performed as detailed [67 (link)]. Human tissue fixed in periodatelysine-paraformaldehyde was blocked, paraffin-embedded, and sectioned at 1μm. Antigen retrieval for α-synuclein and β-amyloid was performed with formic acid treatment for two minutes. Sections were incubated with primary antibodies overnight at 4°C. Antibodies used were α-synuclein (rabbit polyclonal; Chemicon, Temecula, CA; 1:15,000), phosphorylated PHF-tau (AT8; Pierce Endogen, Rockford, IL; 1:2000), and Aβ (4G8; BioLegend, San Diego, CA; 1:100,000). Sections were treated with biotinylated secondary antibody after three PBS washes and labeled with a 3-amino-9-ethylcarbazol HRP substrate kit (Vector Laboratories, Burlingame, CA), counterstained with Gill’s hematoxylin (Vector Laboratories H-3401), and coverslipped with Permount mounting medium (Thermo Scientific, Rockford, IL).

Adams J.W., Alosco M.L., Mez J., Alvarez V.E., Huber B.R., Tripodis Y., Alder C.H., Kubilius C., Cormier K.A., Mathais R., Nicks R., Kelley H.J., Saltiel N., Uretsky M., Nair E., Aytan N., Cherry J.D., Nowinski C.J., Kowall N.W., Goldstein L.E., Dwyer B., Katz D.I., Cantu R.C., Stern R.A., McKee A.C, & Stein T.D. (2020). Association of probable REM sleep behavior disorder with pathology and years of contact sports play in chronic traumatic encephalopathy. Acta neuropathologica, 140(6), 851-862.

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Immunostaining of Brain Sections

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Immunostaining of all brain sections were carried out by University of California-Irvine, Pathology services using Ventana BenchMark Ultra protocols. Neighboring slices were immunostained for Ubiquitin (Cell Marque catalog no. 318A-18, Rocklin, CA, USA) and α-synuclein (EMD Millipore Corporation, lot No. 2985418, Burlington, MA, USA). All IHC stained slides were scanned using the Ventana Roche instrumentation and analyzed using QuPath. Adjacent slices were also immunostained for Aβ plaques and total Tau. All IHC stained slides were scanned using the Ventana Roche instrumentation and analyzed using QuPath [29 (link)].

Mukherjee J., Ladwa R.M., Liang C, & Syed A.U. (2022). Elevated Monoamine Oxidase-A in Anterior Cingulate of Post-Mortem Human Parkinson’s Disease: A Potential Surrogate Biomarker for Lewy Bodies?. Cells, 11(24), 4000.

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Brain Immunostaining for Protein Analysis

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Campoy A.D., Liang C., Ladwa R.M., Patel K.K., Patel I.H, & Mukherjee J. (2021). [18F]Nifene PET/CT Imaging in Mice: Improved Methods and Preliminary Studies of α4β2* Nicotinic Acetylcholinergic Receptors in Transgenic A53T Mouse Model of α-Synucleinopathy and Post-Mortem Human Parkinson’s Disease. Molecules, 26(23), 7360.

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Protein Analysis of Brain Tissue

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Animals were anesthetized by isoflurane inhalation, and the brains were immediately recovered after decapitation and quickly placed in dry ice. Tissue and cell samples were homogenized in a lysis buffer (Ozyme, Saint-Quentin-en-Yvelines, France) or RIPA buffer (Sigma-Aldrich) and analyzed by electrophoresis and protein transfer onto a PVDF membrane (Thermo Fisher Scientific). For all experiments, 30 μg of proteins was used. Membranes were probed with polyclonal antibodies against TH (Millipore) diluted 1:200, anti-active caspase 3 diluted 1:200 (Cell Signaling, Danvers, USA), phosphoTH-Ser31 diluted 1:500, phosphoTH-Ser40 (Ozyme) diluted 1:400, BiP/GRP78 (Sigma-Aldrich) diluted 1:100, DJ-1/PARK7 (Millipore) diluted 1:100, α-synuclein (Sigma-Aldrich) diluted 1:250, anti-H3K27me diluted 1:500 (Diagenode, Ougrée, Belgium) and anti-H3 diluted 1:500 (Abcam). After incubation with Alexia conjugated secondary antibodies (Thermo Fisher Scientific) diluted at 1:1000, the resulting immune complexes were visualized using the ECL chemiluminescence system (Thermo Fischer Scientific). An antibody against α-tubulin (Sigma-Aldrich) or GAPDH (Sigma-Aldrich) diluted at 1:1000, was used as a control to ensure equal protein loading.

Alsharif I., Boukhzar L., Lefranc B., Godefroy D., Aury-Landas J., Rego J.L., Rego J.C., Naudet F., Arabo A., Chagraoui A., Maltête D., Benazzouz A., Baugé C., Leprince J., Elkahloun A.G., Eiden L.E, & Anouar Y. (2020). Cell-penetrating, antioxidant SELENOT mimetic protects dopaminergic neurons and ameliorates motor dysfunction in Parkinson's disease animal models. Redox Biology, 40, 101839.

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Inflammasome Activation in MDMi Cells

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For inflammasome activation experiments, MDMi were primed with 200 ng/ml of ultrapure LPS (E.Coli 0111:B4, Invivogen) for 3 h or 50 μg of S-clamp or F-clamp for 6 h. Cells were washed in after priming to remove residual LPS or S-Clamp and cells were stimulated with conventional NLRP3 inflammasome activators ATP (5 mM, Sigma) and nigericin (10 μM, Invivogen), or fibrillar α-synuclein (10 μM, Proteos), S-Clamp (2–50 μg) or SARS-CoV-2 isolates (MOI 0.1, 1) for the indicated time. For priming studies, MDMis were pre-treated with the NF-kB inhibitor, Bay 11-7082 (3 μM, Sigma), before stimulation with S-clamp and the addition of ATP, nigericin or α-synuclein. For inhibition studies, MCC950 (10 μM), VX-765 (20 μM, Invivogen) and MLN-4760 (1,10 μM, Sigma) were added after the priming step. At the end of treatment, the supernatant was collected and stored at −80 °C until analysis by enzyme-linked immunosorbent assay (ELISA) or western blotting.

Albornoz E.A., Amarilla A.A., Modhiran N., Parker S., Li X.X., Wijesundara D.K., Aguado J., Zamora A.P., McMillan C.L., Liang B., Peng N.Y., Sng J.D., Saima F.T., Fung J.N., Lee J.D., Paramitha D., Parry R., Avumegah M.S., Isaacs A., Lo M.W., Miranda-Chacon Z., Bradshaw D., Salinas-Rebolledo C., Rajapakse N.W., Wolvetang E.J., Munro T.P., Rojas-Fernandez A., Young P.R., Stacey K.J., Khromykh A.A., Chappell K.J., Watterson D, & Woodruff T.M. (2022). SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike protein. Molecular Psychiatry, 28(7), 2878-2893.

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Protein Expression Analysis in SH-SY5Y Cells

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Protein was extracted from SH-SY5Y cells using lysis buffer. For western blot analysis, equal amounts of protein were separated using 12% SDS–PAGE gels and transferred onto PVDF membranes by electroblotting. After blocking with 5% fat-free milk for 1 h, the membranes were incubated with primary antibodies α-synuclein (#SAB4502829, SigmaAldrich, Louis, MO, USA), GPX4 (#ab125066, Abcam, Cambridge, USA), FTH1 (#4393, Cell Signaling Technology, Danvers, USA) overnight at 4 °C and followed by appropriate species of HRP-conjugated secondary antibodies at 37 °C for 1 h. Finally, proteins were visualized by ECL reagents detection and the immunoreactive bands were quantified using the Image J software.

Wang M., Li T., Gao R., Zhang Y, & Han Y. (2023). Identifying the potential genes in alpha synuclein driving ferroptosis of Parkinson’s disease. Scientific Reports, 13, 16893.

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Characterization of TiO2 and ZnO NPs

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The TiO₂ and ZnO nanoparticles were obtained from Nanostructured and Amorphous Materials Inc., Los Alamos, NM, USA.
Phosphate buffered saline (PBS), bovine serum albumin (BSA), water, hydrofluoric acid, silver nitrate, hydrogen tetrachloroaurate (III), ethanol, 2-propanol, β-amyloid fragment 1-40 (βA), and α-synuclein were obtained from Sigma Aldrich. All reagents and solvents used in this study were analytical grades.

Slekiene N., Snitka V., Bruzaite I, & Ramanavicius A. (2022). Influence of TiO2 and ZnO Nanoparticles on α-Synuclein and β-Amyloid Aggregation and Formation of Protein Fibrils. Materials, 15(21), 7664.

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Preparation of α-synuclein and Surfactant Solutions

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α-synuclein (Sigma-Aldrich, Steinheim, Germany) monomers were dissolved in phosphate buffer (50 mM) containing Na₂HPO₄ and NaH₂PO₄ (Sigma-Aldrich, Steinheim, Germany) at 315 µM concentration in order to obtain the stock solution.
Stock solutions of surfactants were prepared by dissolving SDS (≥98.5%, MW 288.38, Sigma-Aldrich, Steinheim, Germany), CTAC (25 wt% solution in water, MW 320.01, Sigma-Aldrich, Steinheim, Germany), or OG (≥98%, MW 292.37, Sigma-Aldrich, Steinheim, Germany) in phosphate buffered saline (PBS), pH 7.4 (10 mM phosphate buffer, 2.7 mM potassium chloride, and 137 mM sodium chloride, Sigma-Aldrich, Steinheim, Germany).

Loureiro J.A., Andrade S., Goderis L., Gomez-Gutierrez R., Soto C., Morales R, & Pereira M.C. (2021). (De)stabilization of Alpha-Synuclein Fibrillary Aggregation by Charged and Uncharged Surfactants. International Journal of Molecular Sciences, 22(22), 12509.

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