Alpha Synuclein Monomers (Type 2)

• Catalog Number: orb1822364
• Category: Proteins
• Description: Human Recombinant Alpha Synuclein Protein Monomers (Type 2)
• Tested applications: In vitro, In vivo, SDS-PAGE, WB
• Reactivity: Human
• Tag: No tag
• Concentration: 2 mg/ml
• Purity: > 95%
• MW: ~14.46 kDa
• Target: Alpha Synuclein
• UniProt ID: P37840
• Protein Sequence: MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA
• Protein Length: Full Length
• Source: Recombinant
• Expression System: E. coli
• Biological Activity: Thioflavin T curve shows less β-sheet aggregation when Type 2 monomers (SPR-316) are seeded with PFFs compared to Type 1 monomers (SPR-321) seeded with PFFs.
• Storage: -80°C
• Buffer/Preservatives: PBS pH 7.4
• Alternative names: Alpha synuclein monomer, Alpha-synuclein monomer,
• Note: For research use only
• Application notes: Certified 95% pure using SDS-PAGE analysis. Low endotoxin < 5 EU/mL @ 2mg/mL.
• Expiration Date: 6 months from date of receipt.

SDS-PAGE of ~14 kDa Human Recombinant Alpha Synuclein Protein Monomer. Lane 1: Molecular Weight Ladder (MW). Lane 2: BSA (2.5 μg). Lane 3: BSA (5 μg). Lane 4: Alpha Synuclein Protein Monomer (2.5 μg). Lane 5: Alpha Synuclein Protein Monomer (5 μg).

Thioflavin T is a fluorescent dye that binds to beta sheet-rich structures, such as those in alpha synuclein fibrils. Upon binding, the emission spectrum of the dye experiences a red-shift and increased fluorescence intensity. Thioflavin T emission curves show increased fluorescence (correlated to alpha synuclein protein aggregation) over time when 10 μM of Type 1 alpha synuclein pre-formed fibrils is combined with 100 μM of Type 1 alpha synuclein monomer, compared to 10 μM of Type 1 alpha synuclein pre-formed fibrils combined with 100 μM Type 2 alpha synuclein monomer. Type 2 fibrils do not seed type 2 monomers (data not shown). Thioflavin T ex = 450 nm, em = 485 nm.

Type 2 monomers are currently undergoing testing in a Real-Time Quaking-Induced Conversion (RT-QuIC) assay. At 10 μg/well there was discrimination between positive and negative CSF samples, and the unseeded reaction occurred later than either the LB BH or the positive CSF sample. This suggests Type 2 monomers could potentially be used as a substrate for alpha synuclein RT-QuIC. Further testing/optimization is underway. LB BH: 10% Lewy body disease; CSF +: CSF from patient with neuropathologically confirmed alpha-synucleinopathy; CSF -: CSF from patient with no evidence of alpha-synuclein deposition at postmortem. Image source: Alison Green, Graham Fairfoul

Evaluation of a-syn toxicity on primary mouse cortical neurons. Mitochondrial dehydrogenase activity reduces yellow MTT to dark blue formazan crystals, a reaction catalyzed in living cells. Cell viability was assessed with an MTT assay and displayed as % of vehicle control (VC). Data are presented as bar graphs and standard deviation.

Evaluation of a-syn toxicity on primary mouse cortical neurons. Lactate dehydrogenase (LDH) is a soluble enzyme present in the cytosol that is released upon cell death. Toxicity was assessed with an LDH assay and displayed as % of vehicle control (VC). Data are presented as bar graphs and standard deviation. For statistical analysis One-way ANOVA followed by Bonferroni post-hoc test (vs VC) was used.