Biological Origin: Mouse. Biological Activity: 100 µM alpha synuclein protein monomer seeded with 10 uM alpha synuclein protein PFF in 25 µM Thioflavin T (PBS pH 7.4, 100 µl reaction volume) generated an increased fluorescence intensity after incubation at 37C with shaking at 600 rpm. Fluorescence was measured by excitation at 450 nm and emission at 485 nm on a Molecular Devices Gemini XPS microplate reader. Application Notes: Certified >95% pure using SDS-PAGE analysis. Low endotoxin <5 EU/mL 2mg/mL
C57/BL6 mice were injected with sonicated recombinant mouse alpha synuclein monomers or fibrils at 8 weeks of age. Mice were unilaterally injected in the dorsal striatum (bregma AP + 0.2 mm, L +/1 2.0 mm, V - 3.0 mm) and sacrificed 30 days post-injection. (A) 1.25 uL mouse alpha synuclein monomers. (B) 2.5 uL mouse alpha synuclein monomers. (C) 2.5 ug alpha synuclein PFFs. (C) 5 ug alpha synuclein PFFs Inset: PBS (negative control). Primary antibody: Anti-Alpha Synuclein pSer129 at 1:10 000. Secondary antibody: anti-rabbit HRP. Mice injected with PFF displayed alpha synuclein staining in the striatum and cortex and contralateral to the injection site.
Type 1 alpha synuclein pre-formed fibrils seed the formation of new alpha synuclein fibrils from the pool of alpha synuclein monomers. 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 alpha synuclein monomer, as compared to Type 1 alpha synuclein pre-formed fibrils or alpha synuclein monomer alone. Thioflavin T ex = 450 nm, em = 485 nm.
SDS-PAGE of ~14 kDa Mouse Recombinant Alpha Synuclein Protein Monomer. Lane 1: Molecular Weight Ladder (MW). Lane 2: Alpha Synuclein Protein Monomer (2 µg).
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