Active alpha synuclein preformed fibrils (GTX17669-pro) seed the formation of new alpha synuclein fibrils from the pool of alpha synuclein monomers (GTX17668-pro). 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 nM of active alpha synuclein preformed fibrils (GTX17669-pro) is combined with 100 μM of alpha synuclein monomer (GTX17668-pro), as compared to active alpha synuclein preformed fibrils (GTX17669-pro) alone and alpha synuclein monomer (GTX17668-pro) alone. Thioflavin T ex = 450 nm, em = 485 nm.
Immunohistochemistry analysis of rat brain injected with active human alpha synuclein PFFs (GTX17669-pro). Species: Female Sprague-Dawley Rat. Rat was injected with 2μL active human alpha synuclein PFFs (GTX17669-pro) in each of 2 injection sites: AP+1.6, ML+2.4, DV-4.2 from skull; and AP-1.4, ML+0.2, DV-2.8 from skull. 30-days post-injection. Fixation: Saline perfusion followed by 4% PFA fixation for 48 hrs. Secondary Antibody: Biotin-SP Donkey Anti-Rabbit IgG (H+L) at 1:500 for 2 hours in cold room with shaking. ABC signal amplification, DAB staining. Magnification: 20X. Alpha synuclein pathology is seen in the striatum close to an injection site.
Primary rat hippocampal neurons show lewy body inclusion formation when treated with active Alpha Synuclein Protein Preformed Fibrils (GTX17669-pro) at 4 μg/ml (D-F), but not when treated with control Alpha Synuclein Protein Preformed Fibrils (GTX17667-pro) at 4 μg/ml (A-C). Tissue: Primary hippocampal neurons. Species: Sprague-Dawley rat. Fixation: 4% formaldehyde from PFA. Primary Antibody: Mouse anti-pSer129 Antibody at 1:1000 24 hours at 4ºC. Secondary Antibody: FITC Goat Anti-Mouse (green) at 1:700 for 1 hours at RT. Counterstain: Hoechst (blue) nuclear stain at 1:4000 for 1 hour at RT. Localization: Lewy body inclusions. Magnification: 20x.
SDS-PAGE of ~14 kDa active Human Recombinant Alpha Synuclein Protein Preformed Fibrils (GTX17669-pro). Lane 1: Molecular Weight Ladder (MW). Lane 2: active Alpha Synuclein Protein Preformed Fibrils (GTX17669-pro).
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