Endogenous alpha-synuclein phosphorylation. 100 µM alpha synuclein protein monomer (GTX17670-pro) seeded with 10 nM alpha synuclein protein PFF (GTX17671-pro) in 25 µM Thioflavin T (PBS pH 7.4, 100 µl reaction volume) generated an increased fluorescence intensity after incubation at 37?C with shaking at 600 rpm for 24 hours. Fluorescence was measured by excitation at 450 nm and emission at 485 nm on a Molecular Devices Gemini XPS microplate reader.
Active alpha synuclein preformed fibrils (GTX17671-pro) seed the formation of new alpha synuclein fibrils from the pool of alpha synuclein monomers (GTX17670-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 (GTX17671-pro) is combined with 100 μM of alpha synuclein monomer (GTX17670-pro), as compared to active alpha synuclein preformed fibrils (GTX17671-pro) or alpha Synuclein monomer (GTX17670-pro) alone. Thioflavin T ex = 450 nm, em = 485 nm.
Primary rat hippocampal neurons (DIV16) show lewy body inclusion formation and loss of cells when treated with active mouse Alpha Synuclein Protein Preformed Fibrils (GTX17671-pro) at 4 μg/ml (D-F) on DVI2, but not when treated with a control (A-C). Tissue: Primary hippocampal neurons. Species: Sprague-Dawley rat. Fixation: 3% formaldehyde from PFA for 20 min. Blocker: 1:1 PBS:LiCOR Odyssey Block (LiCOR, 927-40010) and 30 mL/mL of 0.1% triton-X 100 for 30 min. Primary Antibody: Mouse anti-pSer129 Antibody (1:1000) and Rabbit anti-pSer129 (1:800) for 24 hours at 4°C. Secondary Antibody: ATTO 546 Donkey Anti-Mouse (1:700) and ATTO 488 Donkey Anti-Rabbit (1:700) for 1 hour at RT (composite green). Counterstain: Hoechst (blue) nuclear stain at 1:3000 for 1 hour at RT. Localization: Lewy body incluscions. Magnification: 20x.
TEM of active mouse alpha synuclein preformed fibrils (GTX17671-pro). Fibrils were sonicated and image was taken at 100kx magnification.
TEM of active mouse alpha synuclein preformed fibrils (GTX17671-pro). Image was taken at 100kx magnification.
Immunohistochemistry analysis of rat brain injected with active mouse alpha synuclein PFFs (GTX17671-pro). Species: Female Sprague-Dawley Rat. Rat was injected with 2μL active mouse alpha synuclein PFFs (GTX17671-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 periform/insular cortex and the cingulate cortex on both the same (ipsi) and opposite (contra) sides as the injection sites.
SDS-PAGE of ~14 kDa active Mouse alpha Synuclein protein (active, fibrils) (GTX17671-pro). Lane 1: Molecular Weight Ladder (MW). Lane 2: active Mouse alpha Synuclein protein (active, fibrils) (2 μg) (GTX17671-pro).
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