Alpha Synuclein Pre-formed Fibrils (Type 1)

• Catalog Number: orb1822362
• Category: Proteins
• Description: Mouse Recombinant Alpha Synuclein Pre-formed Fibrils (Type 1)
• Tested applications: In vitro, In vivo, SDS-PAGE, WB
• Reactivity: Mouse
• Tag: No tag
• Concentration: 2 mg/ml or 5 mg/ml
• Purity: > 95%
• MW: ~14.46 kDa
• Target: Alpha Synuclein
• UniProt ID: O55042
• Protein Sequence: MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVTTVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGNIAA ATGFVKKDQM GKGEEGYPQE GILEDMPVDP GSEAYEMPSE EGYQDYEPEA
• Protein Length: Full Length
• Source: Recombinant
• Expression System: E. coli
• Biological Activity: Endogenous alpha-synuclein phosphorylation. 100 µM alpha synuclein protein monomer (SPR-323) seeded with 10 uM alpha synuclein protein PFF (SPR-324) 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. 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: Alpha synuclein PFFs, Alpha synuclein PFF, Alpha s
• 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 and loss of cells when treated with Type 1 mouse Alpha Synuclein Protein Pre-formed Fibrils 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) 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 inclusions. Magnification: 20x.

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. (D) 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.

C57/BL6 mice were injected with 5 ug sonicated mouse recombinant alpha synuclein PFFs 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) contralateral cortex. (B) ipsilateral cortex. (C) contralateral striatum. (D) ipsilateral striatum. 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.

Primary mouse hippocampal neurons treated with 100 nM sonicated mouse alpha synuclein PFFs (A). Phosphorylated alpha synuclein (detected with pSer129 antibody) was visible in perinucleus and neurites compared to untreated control (B).

TEM of Type 1 mouse alpha synuclein Pre-formed Fibrils. Image was taken at 100kx magnification.

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.

TEM of Type 1 mouse alpha synuclein Pre-formed Fibrils. Fibrils were sonicated and image was taken at 100kx magnification.

SDS-PAGE of ~14 kDa Type 1 Mouse Recombinant Alpha Synuclein Protein Pre-formed Fibrils. Lane 1: Molecular Weight Ladder (MW). Lane 2: Type 1 Alpha Synuclein Protein Pre-formed Fibrils (2 μg).

Immunohistochemistry analysis of rat brain injected with Type 1 mouse alpha synuclein PFFs. Species: Female Sprague-Dawley Rat. Rat was injected with 16μg Type 1 mouse alpha synuclein PFFs 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. Primary antibody: rabbit monoclonal anti-pSer129 alpha synuclein. 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.