Product Name | Alpha Synuclein Protein | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Description |
Human Recombinant Alpha Synuclein Pre-formed Fibrils (Type 2) |
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Applications | WB, SDS-PAGE, In vivo assay, In vitro assay | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Concentration | 2 mg/mL | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Conjugates |
No tag
StreptavidinProperties:
BiotinProperties: HRP (Horseradish peroxidase)Properties:
AP (Alkaline Phosphatase)Properties:
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Nature | Recombinant | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Species | Human | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Expression System | E. coli | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Amino Acid Sequence | MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Purity | >95% | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Other Resources | Sonication Protocol | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Protein Length | Full Length | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Protein Size | ~14.46 kDa | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Biological Activity | Does not induce Lewy body inclusion formation in Sprague-Dawley rat primary hippocampal neurons. Thioflavin T emission curve shows only a small increase in fluorescence (indicative of alpha synuclein aggregation) when Type 2 alpha synuclein PFFs (SPR-317) are combined with alpha synuclein monomers (SPR-321 or SPR-316). Certain biological activities in other neuronal cells cannot be ruled out. Researchers should test compatibility prior to use. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Field of Use | Not for use in humans. Not for use in diagnostics or therapeutics. For in vitro research use only. |
Storage Buffer | PBS pH 7.4 |
Storage Temperature | -80ºC |
Shipping Temperature | Dry Ice. Shipping note: Product will be shipped separately from other products purchased in the same order. |
Purification | Ion-exchange Purified |
Cite This Product | Human Recombinant Alpha Synuclein Protein (StressMarq Biosciences, Canada, Cat # SPR-317) |
Certificate of Analysis | Certified 95% pure using SDS-PAGE analysis. Made with low endotoxin monomer. |
Other Relevant Information | For best results, sonicate immediately prior to use. Refer to the Neurodegenerative Protein Handling Instructions on our website, or the product datasheet for further information. Monomer source is catalog# SPR-316. |
Alternative Names | Alpha synuclein PFFs, Alpha synuclein PFF, Alpha synuclein aggregates, 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, Parkinson disease familial 1 Protein |
Research Areas | Alzheimer's Disease, Neurodegeneration, Neuroscience, Parkinson's Disease, Synuclein, Tangles & Tau, Multiple System Atrophy |
Cellular Localization | Cytoplasm, Membrane, Nucleus |
Accession Number | NP_000336.1 |
Gene ID | 6622 |
Swiss Prot | P37840 |
Scientific Background | Alpha-Synuclein (SNCA) is expressed predominantly in the brain, where it is concentrated in presynaptic nerve terminals (1). Alpha-synuclein is highly expressed in the mitochondria of the olfactory bulb, hippocampus, striatum and thalamus (2). Functionally, it has been shown to significantly interact with tubulin (3), and may serve as a potential microtubule-associated protein. It has also been found to be essential for normal development of the cognitive functions; inactivation may lead to impaired spatial learning and working memory (4). SNCA fibrillar aggregates represent the major non A-beta component of Alzheimers disease amyloid plaque, and a major component of Lewy body inclusions, and Parkinson's disease. Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive accumulation in selected neurons of protein inclusions containing alpha-synuclein and ubiquitin (5, 6). |
References |
1. “Genetics Home Reference: SNCA”. US National Library of Medicine. (2013). 2. Zhang L., et al. (2008) Brain Res. 1244: 40-52. 3. Alim M.A., et al. (2002) J Biol Chem. 277(3): 2112-2117. 4. Kokhan V.S., Afanasyeva M.A., Van'kin G. (2012) Behav. Brain. Res. 231(1): 226-230. 5. Spillantini M.G., et al. (1997) Nature. 388(6645): 839-840. 6. Mezey E., et al. (1998) Nat Med. 4(7): 755-757. |
Primary rat hippocampal neurons show lewy body inclusion formation when treated with Type 1 Alpha Synuclein Pre-formed Fibrils (SPR-322) at 4 µg/ml (D-F), but not when treated with Type 2 Alpha Synuclein Pre-formed Fibrils (SPR-317) at 4 µg/ml (A-C). Tissue: Primary hippocampal neurons. Species: Sprague-Dawley rat. Fixation: 4% formaldehyde made from PFA. Primary Antibody: Mouse anti-pSer129 Antibody at 1:1000 24 hours at 4°C; Secondary Antibody: FITC Goat Anti-Mouse (green) at 1:700 for 1 hours at RT (B, E). Counterstain: Hoechst (blue) nuclear stain at 1:4000 for 1 hour at RT (A,D). Localization: Lewy body inclusions. Magnification: 20x. C is A and B merged and F is D and E merged.
Type 1 alpha synuclein Pre-formed fibrils (SPR-322) seed the formation of new alpha synuclein fibrils from the pool of alpha synuclein monomers (SPR-321). 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 (SPR-322) is combined with 100 µM of alpha synuclein monomer (SPR-321), as compared to when 10 µM of Type 2 alpha synuclein Pre-formed fibrils (SPR-317) is combined with 100 µM of alpha synuclein monomer (SPR-321) or 100 µM of alpha Synuclein monomer (SPR-316). Thioflavin T ex = 450 nm, em = 485 nm. Note: We use molecular weight of 14.46 kDa for both alpha synuclein monomer and fibril in calculations. We load 100µL/well for Thioflavin T assay so 100 µM is 144.6µg/well and 10 µM is 14.46 µg/well.
Toxicity results comparing Active Human Recombinant Alpha Synuclein Pre-formed Fibrils (Type 2) (Catalog No. SPR-317) and Active Human Recombinant Alpha Synuclein Pre-formed Fibrils (Type 1) (Catalog No. SPR-322). 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.
Evaluation of a-syn toxicity on primary mouse cortical neurons. Lactate dehydrogenase (LDH) is a soluble enzyme present in the cytosol that is released upon cell death. Toxicity was assessed with an LDH assay and displayed as % of vehicle control (VC). Data are presented as bar graphs and standard deviation. For statistical analysis One-way ANOVA followed by Bonferroni post-hoc test (vs VC) was used. *** p<0.001. Treatment with Type 2 PFFs did not significantly impact LDH release. Data courtesy of QPS.
Evaluation of a-syn toxicity on primary mouse cortical neurons. Mitochondrial dehydrogenase activity reduces yellow MTT to dark blue formazan crystals, a reaction catalyzed in living cells. Cell viability was assessed with an MTT assay and displayed as % of vehicle control (VC). Data are presented as bar graphs and standard deviation. For statistical analysis One-way ANOVA followed by Bonferroni post-hoc test (vs VC) was used. ** p<0.01, *** p<0.001. Treatment with Type 2 PFFs reduced cell viability (p<0.001). Data courtesy of QPS.
UV-CD data suggests StressMarq’s Alpha Synuclein Pre-formed Fibrils (PFF), Type 1 (cat#SPR-322) and Type 2 (cat#SPR-317) both have a high beta sheet/turn content, yet do have small secondary structure differences. StressMarq’s Alpha Synuclein Monomers (cat#SPR-316) show a strong negative signal at 200 nm indicative of a disordered protein state (low secondary structure content). For this experiment, pre-formed fibrils (PFF) were subjected to 10 cycles of sonication prior to UV-CD to ensure solubility prior to measurement.
TEM of Type 2 Alpha Synuclein Fibrils (SPR-317) after 30 cycles of high frequency sonication using a Bioruptor Pico. Negative stain transmission electron microscopy (TEM) images acquired at 80 Kv on carbon coated 400 mesh copper grids using phosphotungstic acid and uranyl acetate stain. Scale bar = 200 nm.
StressMarq Biosciences :
Based on validation through cited publications.
Yu Zhang :
How much is it in China
Patricia Thomson :
Please contact one of our Chinese distribitors for pricing. http://www.stressmarq.com/company/distributors/