PRODUCT
Summary
Active Mouse Recombinant Alpha Synuclein Pre-formed Fibrils (Type 1)
APPLICATION
Application Note
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 37ºC 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.
*For best results, sonicate immediately prior to use.
PROPERTIES
Form
Liquid
Buffer
PBS
Preservative
No preservative
Storage
Store as concentrated solution. Aliquot and store at -20ºC or below. Avoid freeze-thaw cycles.
Concentration
Batch dependent (Please refer to the vial label for the specific concentration.)
Region/Sequence
Full-length without tagged; MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVTTVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGNIAA ATGFVKKDQM GKGEEGYPQE GILEDMPVDP GSEAYEMPSE EGYQDYEPEA
Expression System
E. coli
Purification
Purified by ion-exchange chromatography
Purity
> 95% by SDS-PAGE
Conjugation
Unconjugated
Note
For laboratory use only. Not for any clinical, therapeutic, or diagnostic use in humans or animals. Not for animal or human consumption.
TARGET
Synonyms
synuclein, alpha , synuclein, alpha , NACP , alpha-Syn , alphaSYN
Database
Research Area
DATA IMAGES
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GTX17671-pro Functional Assay Image
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.
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GTX17671-pro Functional Assay Image
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 4ºC. 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.
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GTX17671-pro Image
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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GTX17671-pro Image
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.
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REFERENCE
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REVIEW
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