Alpha Synuclein Pre-formed Fibrils (Type 1)

Article Name:	Alpha Synuclein Pre-formed Fibrils (Type 1)
Biozol Catalog Number:	BYT-ORB1822365
Supplier Catalog Number:	orb1822365
Alternative Catalog Number:	BYT-ORB1822365-100
Manufacturer:	Biorbyt
Category:	Proteine/Peptide
Application:	In vitro, In vivo, SDS-PAGE, WB
Species Reactivity:	Human
Alternative Names:	Alpha synuclein PFFs, Alpha synuclein aggregates, Alpha synuclein PFF, Alpha synuclein protein aggregates, Alpha synuclein aggregates, Alpha-synuclein protein, Non-A beta component of AD amyloid protein, Non-A4 component of amyloid precursor protein, NACP protein, SNCA protein, NACP protein, PARK1 protein, SYN protein, Parkinsons disease familial 1 Protein, Alpha synuclein protein seed

Human Recombinant Alpha Synuclein Pre-formed Fibrils (Type 1)

Concentration:	2 mg/ml or 5 mg/ml
Molecular Weight:	~14.46 kDa
UniProt:	P37840
Buffer:	PBS
Purity:	>95%
Sequence:	MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA
Target:	Alpha Synuclein

Application Notes:

Biological Activity: Endogenous alpha-synuclein phosphorylation. 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 a fluorescence intensity of 13,000 Relative Fluorescence Units 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

	Primary rat hippocampal neurons show lewy body inclusion formation when treated with Type 1 Alpha Synuclein Protein Pre-formed Fibrils at 4 µg/ml (D-F), but not when treated with Type 2 Alpha Synuclein Protein Pre-formed Fibrils 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 4C. 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.
	ATTO633 fluorescently-labelled alpha synuclein PFFs were taken up, transported into the soma, and induced alpha synuclein aggregation in mouse neurocortical primary cells. (A) Neurites filled with fluorescently-labelled alpha synuclein seeds in a microfluidic co-culture system after 24 hours. (B) Alpha synuclein seeds within the soma and neurites of mouse neurocortical primary cells after 24 hours.
	Confocal imaging shows NeuN+ (mature) primary cortical neurons filled with ATTO633 fluorescently-labelled alpha synuclein PFFs. ATTO-633 alpha synuclein PFFs seed endogenous alpha synculein phosphorylation after 7 days.
	TEM of Type 1 Alpha Synuclein Pre-formed Fibrils (PFFs)
	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 alone and alpha synuclein monomer alone. Thioflavin T ex = 450 nm, em = 485 nm.
	TEM of Type 1 Alpha Synuclein Pre-formed Fibrils (PFFs)
	SDS-PAGE of ~14 kDa Type 1 Human Recombinant Alpha Synuclein Protein Pre-formed Fibrils. Lane 1: Molecular Weight Ladder (MW). Lane 2: Type 1 Alpha Synuclein Protein Pre-formed Fibrils.
	Toxicity results comparing Active Human Recombinant Alpha Synuclein Pre-formed Fibrils (Type 2) and Active Human Recombinant Alpha Synuclein Pre-formed Fibrils (Type 1). Data was graphed after live cell imaging results were obtained using the following procedure: After 8 days in vitro, primary rat mixed cortical neuron cells were washed with 1X PBS and treated with 500 µg/ml of Type 1 and Type 2 Alpha Synuclein Proteins for 20 hours at 37?C. Following treatements, cells were washed with 2X PBS and incubated with a staining solution (2.0 µM Cell Event + 2.5 µM Ethidium homodimer + 2.5 µg/ml Hoechst 33342 in sterile HBSS) for 30 minutes at 37?C. The addition of the Type 2 Alpha Synuclein Proteins resulted in a significant increase in cell death.
	Immunohistochemistry analysis of rat brain injected with Type 1 human alpha synuclein PFFs. Species: Female Sprague-Dawley Rat. Rat was injected with 16µg Type 1 human 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 striatum close to an injection site.