Alpha Synuclein A53T Mutant Pre-formed Fibrils (Type 1)

• Catalog Number: orb1822360
• Category: Proteins
• Description: Human Recombinant A53T Mutant Alpha Synuclein Protein Pre-formed Fibrils (Type 1)
• 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 A53T Mutant
• UniProt ID: P37840
• Protein Sequence: MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVTTVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA
• Protein Length: Full Length
• Source: Recombinant
• Expression System: E. coli
• Biological Activity: 100 µM A53T alpha synuclein protein monomer (SPR-325) seeded with 10 uM A53T alpha synuclein protein PFF (SPR-326) in 25 µM Thioflavin T (PBS pH 7.4, 100 µl reaction volume) generated a fluorescence intensity of 28 000 Relative Fluorescence Units after incubation at 37°C with shaking at 600 rpm for 56 hours. Fluorescence was measured by excitation at 450 nm and emission at 485 nm on a Molecular Devices Gemini XPS microplate reader.
• Storage: -80°C
• Buffer/Preservatives: PBS pH 7.4
• Alternative names: A53T mutant alpha synuclein, A53T mutated SNCA, A5
• 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.

Primary rat hippocampal neurons show lewy body inclusion formation when treated with A53T mutant Alpha Synuclein Protein Pre-formed Fibrils (B) but not when treated with a media control (A). Tissue: Primary hippocampal neurons. Species: Sprague-Dawley rat. Primary Antibody: Rabbit anti-pSer129 Antibody. Fibrils were diluted to 1 ug/uL in neuronal media consisting of B27, Glutamax, penicillin/strip, and neurobasalA and sonicated for 1 hour in a water bath. The sonicated stock was then used to achieve the final concentration of 1 ug/mL in the wells.

TEM of A53T alpha synuclein Pre-formed Fibrils

TEM of A53T alpha synuclein Pre-formed Fibrils

A53T alpha synuclein monomers and fibrils are well recognized by homogeneous time-resolved fluorescence (HTRF) total alpha synuclein assay.

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 a limited increase in fluorescence (correlated to alpha synuclein aggregation) over time in A53T alpha synuclein monomers. A much greater increase in fluorescence is seen when 100 μM monomer is combined with 10 μM of fibrils as the fibrils seed the formation of new fibrils from the pool of active monomers. Thioflavin T ex = 450 nm, em = 485 nm.

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.

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.

A53T alpha synuclein monomers and fibrils are well recognized by homogeneous time-resolved fluorescence (HTRF) alpha synuclein aggregation assay.