Alpha Synuclein pSer129 Pre-Formed Fibrils

Name: Alpha Synuclein pSer129 Pre-Formed Fibrils
SKU: SPR-521
Availability: InStock

Human Recombinant Alpha Synuclein Pre-formed Fibrils phosphorylated at position 129

Catalog No. SPR-521

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Expression System	E. coli
Tags	No tag

SKU: SPR-521 Category: Proteins

Sedimentation assay of Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521)

Dot Blot of Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521)

Mass Spec Bar Graph of Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521)

Thioflavin T assay of Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521)

TEM of Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521)

AFM of Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521)

SDS-Page of Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521)

Product Name Alpha Synuclein pSer129 Pre-Formed Fibrils

Description

Human Recombinant Alpha Synuclein Pre-formed Fibrils phosphorylated at position 129

Applications WB, SDS-PAGE, In vivo assay, In vitro assay

Concentration 2 mg/ml or 5 mg/ml

Conjugates

No tag

Dylight 488

Overview:

High fluorescence yield
High photostability
Less pH-sensitive
Excellent batch-to-batch reproducibility
Stringently QC tested
Molecular weight: 1011 g/mol

Dylight 488 Datasheet

Dylight 488 Fluorophore Absorption and Emission Spectrum

Optical Properties:

λ_ex = 493 nm

λ_em = 518 nm

ε_max = 7.0×10⁴

Laser = 488 nm

APC/Cy7

Overview:

High quantum yield
Excellent batch-to-batch reproducibility
Stringently QC tested

APC-Cy7 Datasheet

ACP-Cy7 Fluorophore Absorption and Emission Spectrum

Optical Properties:

λ_ex = 652 nm

λ_em = 790 nm

Laser = 594 or 633 nm

Dylight 350

Overview:

High fluorescence intensity
High photostability
Less pH-sensitive
Excellent solubility in water
Stringently QC tested
Excellent batch-to-batch reproducibility
Molecular weight: 874 g/mol

Dylight 350 Datasheet

Dylight 350 Fluorophore Absorption and Emission Spectrum

Optical Properties:

λ_ex = 353 nm

λ_em = 432 nm

ε_max = 1.5×10⁴

Dylight 405

Overview:

High fluorescence intensity
High photostability
Less pH-sensitive
Excellent batch-to-batch reproducibility
Stringently QC tested
Molecular weight: 793 g/mol

Dylight 405 Datasheet

Dylight 405 Fluorophore Absorption and Emission Spectrum

Optical Properties:

λ_ex = 400 nm

λ_em = 420 nm

ε_max = 3.0×10⁴

Laser = 405 nm

Dylight 594

Overview:

High fluorescence yield
High photostability
Less pH-sensitive
Excellent batch-to-batch reproducibility
Stringently QC tested
Molecular weight: 1078 g/mol

Dylight 594 Datasheet

Dylight 594 Fluorophore Absorption and Emission Spectrum

Optical Properties:

λ_ex = 593 nm

λ_em = 618 nm

ε_max = 8.0×10⁴

Laser = 526 nm

Dylight 633

Overview:

High fluorescence yield
High photostability
Less pH-sensitive
Excellent batch-to-batch reproducibility
Stringently QC tested
Molecular weight: 1066 g/mol

Dylight 633 Datasheet

Dylight 633 Fluorophore Absorption and Emission Spectrum

Optical Properties:

λ_ex = 638 nm

λ_em = 658 nm

ε_max = 1.7×10⁵

Laser = 633 nm

PerCP

Overview:

Peridinin-Chlorophyll-Protein Complex
Small phycobiliprotein
Isolated from red algae
Large stokes shift (195 nm)
Molecular Weight: 35 kDa

PerCP Datasheet

PerCP Fluorophore Absorption and Emission Spectrum

Optical Properties:

λ_ex = 482 nm

λ_em = 677 nm

ε_max = 1.96 x 10⁶

Laser = 488 nm

PE/ATTO 594

PE/ATTO 594 is a tandem conjugate, where PE is excited at 535 nm and transfers energy to ATTO 594 via FRET (fluorescence resonance energy transfer), which emits at 627 nm.

Overview:

High fluorescence yield
High photostability
Very hydrophilic
Excellent solubility in water
Very little aggregation

PE/ATTO 594 Datasheet

PE-ATTO 594 Fluorophore Conjugate Excitation and Emission Spectra

Optical Properties:

λ_ex = 535 nm

λ_em = 627 nm

Laser = 488 to 561 nm

FITC (Fluorescein)

Overview:

Excellent fluorescence quantum yield
High rate of photobleaching
Good solubility in water
Broad emission spectrum
pH dependent spectra
Molecular formula: C₂₀H₁₂O₅
Molar mass: 332.3 g/mol

FITC-Fluorescent-conjugate

FITC Fluorescein Fluorophore Excitation and Emission Spectra

Optical Properties:

λ_ex = 494 nm

λ_em = 520 nm

ε_max = 7.3×10⁴

Φ_f = 0.92

τ_fl = 5.0 ns

Brightness = 67.2

Laser = 488 nm

Filter set = FITC

ATTO 700

Overview:

High fluorescence yield
Excellent thermal and photostability
Quenched by electron donors
Very hydrophilic
Good solubility in polar solvents
Zwitterionic dye
Molar Mass: 575 g/mol

ATTO 700 Datasheet

ATTO 700 Fluorophore Absorption and Emission Spectrum

Optical Properties:

λ_ex = 700 nm

λ_em = 719 nm

ε_max = 1.25×10⁵

Φ_f = 0.25

τ_fl = 1.6 ns

Brightness = 31.3

Laser = 676 nm

Filter set = Cy®5.5

ATTO 680

Overview:

High fluorescence yield
Excellent thermal and photostability
Quenched by electron donors
Very hydrophilic
Good solubility in polar solvents
Zwitterionic dye
Molar Mass: 631 g/mol

ATTO 680 Datasheet

ATTO 680 Fluorophore Absorption and Emission Spectrum

Optical Properties:

λ_ex = 680 nm

λ_em = 700 nm

ε_max = 1.25×10⁵

Φ_f = 0.30

τ_fl = 1.7 ns

Brightness = 37.5

Laser = 633 – 676 nm

Filter set = Cy®5.5

ATTO 655

Overview:

High fluorescence yield
High thermal and photostability
Excellent ozone resistance
Quenched by electron donors
Very hydrophilic
Good solubility in polar solvents
Zwitterionic dye
Molar Mass: 634 g/mol

ATTO 655 Datasheet

ATTO 655 Fluorophore Absorption and Emission Spectrum

Optical Properties:

λ_ex = 663 nm

λ_em = 684 nm

ε_max = 1.25×10⁵

Φ_f = 0.30

τ_fl = 1.8 ns

Brightness = 37.5

Laser = 633 – 647 nm

Filter set = Cy®5

ATTO 633

Overview:

High fluorescence yield
High thermal and photostability
Moderately hydrophilic
Good solubility in polar solvents
Stable at pH 4 – 11
Cationic dye, perchlorate salt
Molar Mass: 652.2 g/mol

ATTO 633 Datasheet

ATTO 633 Fluorophore Absorption and Emission Spectrum

Optical Properties:

λ_ex = 629 nm

λ_em = 657 nm

ε_max = 1.3×10⁵

Φ_f = 0.64

τ_fl = 3.2 ns

Brightness = 83.2

Laser = 633 nm

Filter set = Cy®5

ATTO 594

Overview:

High fluorescence yield
High photostability
Very hydrophilic
Excellent solubility in water
Very little aggregation
New dye with net charge of -1
Molar Mass: 1137 g/mol

ATTO 594 Datasheet

ATTO 594 Fluorophore Excitation and Emission Spectrum

Optical Properties:

λ_ex = 601 nm

λ_em = 627 nm

ε_max = 1.2×10⁵

Φ_f = 0.85

τ_fl = 3.5 ns

Brightness = 102

Laser = 594 nm

Filter set = Texas Red®

ATTO 565

Overview:

High fluorescence yield
High thermal and photostability
Good solubility in polar solvents
Excellent solubility in water
Very little aggregation
Rhodamine dye derivative
Molar Mass: 611 g/mol

ATTO 565 Datasheet

ATTO 565 Fluorophore Excitation and Emission Spectra

Optical Properties:

λ_ex = 563 nm

λ_em = 592 nm

ε_max = 1.2×10⁵

Φ_f = 0.9

τ_fl = 3.4 n

Brightness = 10

Laser = 532 nm

Filter set = TRITC

ATTO 488

Overview:

High fluorescence yield
High photostability
Very hydrophilic
Excellent solubility in water
Very little aggregation
New dye with net charge of -1
Molar Mass: 804 g/mol

ATTO 488 Datasheet

ATTO 488 Fluorophore Excitation and Emission Spectra

Optical Properties:

λ_ex = 501 nm

λ_em = 523 nm

ε_max = 9.0×10⁴

Φ_f = 0.80

τ_fl = 4.1 ns

Brightness = 72

Laser = 488 nm

Filter set = FITC

ATTO 390

Overview:

High fluorescence yield
Large Stokes-shift (89 nm)
Good photostability
Moderately hydrophilic
Good solubility in polar solvents
Coumarin derivate, uncharged
Low molar mass: 343.42 g/mol

ATTO 390 Datasheet

ATTO 390 Fluorescent Dye Excitation and Emission Spectra

Optical Properties:

λ_ex = 390 nm

λ_em = 479 nm

ε_max = 2.4×10⁴

Φ_f = 0.90

τ_fl = 5.0 ns

Brightness = 21.6

Laser = 365 or 405 nm

APC (Allophycocyanin)

Overview:

High quantum yield
Large phycobiliprotein
6 chromophores per molecule
Isolated from red algae
Molecular Weight: 105 kDa

APC Datasheet

APC Fluorophore Absorption and Emission Spectrum

Optical Properties:

λ_ex = 650 nm

λ_em = 660 nm

ε_max = 7.0×10⁵

Φ_f = 0.68

Brightness = 476

Laser = 594 or 633 nm

Filter set = Cy®5

Streptavidin

Properties:

Homo-tetrameric protein purified from Streptomyces avidinii which binds four biotin molecules with extremely high affinity
Molecular weight: 53 kDa
Formula: C₁₀H₁₆N₂O₃S
Applications: Western blot, immunohistochemistry, and ELISA

Streptavidin Datasheet

Biotin

Properties:

Binds tetrameric avidin proteins including Streptavidin and neuravidin with very high affinity
Molar mass: 244.31 g/mol
Formula: C₁₀H₁₆N₂O₃S
Applications: Western blot, immunohistochemistry, and ELISA

Biotin Datasheet

HRP (Horseradish peroxidase)

Properties:

Enzymatic activity is used to amplify weak signals and increase visibility of a target
Readily combines with hydrogen peroxide (H₂O₂) to form HRP-H₂O₂ complex which can oxidize various hydrogen donors
Catalyzes the conversion of:
- Chromogenic substrates (e.g. TMB, DAB, ABTS) into coloured products
- Chemiluminescent substrates (e.g. luminol and isoluminol) into light emitting products via enhanced chemiluminescence (ECL)
- Fluorogenic substrates (e.g. tyramine, homovanillic acid, and 4-hydroxyphenyl acetic acid) into fluorescent products
High turnover rate enables rapid generation of a strong signal
44 kDa glycoprotein
Extinction coefficient: 100 (403 nm)
Applications: Western blot, immunohistochemistry, and ELISA

HRP Datasheet

AP (Alkaline Phosphatase)

Properties:

Broad enzymatic activity for phosphate esters of alcohols, amines, pyrophosphate, and phenols
Commonly used to dephosphorylate the 5’-termini of DNA and RNA to prevent self-ligation
Catalyzes the conversion of:
- Chromogenic substrates (e.g. pNPP, naphthol AS-TR phosphate, BCIP) into coloured products
- Fluorogenic substrates (e.g. 4-methylumbelliferyl phosphate) into fluorescent products
Molecular weight: 140 kDa
Applications: Western blot, immunohistochemistry, and ELISA

AP Datasheet

R-PE (R-Phycoerythrin)

Overview:

Broad excitation spectrum
High quantum yield
Photostable
Member of the phycobiliprotein family
Isolated from red algae
Excellent solubility in water
Molecular Weight: 250 kDa

R-PE Datasheet

R-PE Fluorophore Excitation and Emission Spectra

Optical Properties:

λ_ex = 565 nm

λ_em = 575 nm

ε_max = 2.0×10⁶

Φ_f = 0.84

Brightness = 1.68 x 10³

Laser = 488 to 561 nm

Filter set = TRITC

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 140 AA

Protein Size ~14.46 kDa

Field of Use Not for use in humans. Not for use in diagnostics or therapeutics. For in vitro research use only.

Properties

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 pSer129 Pre-Formed Fibrils (StressMarq Biosciences Inc., Victoria BC CANADA, Catalog # SPR-521)
Certificate of Analysis	Protein certified >95% pure using SDS-PAGE analysis. Low endotoxin <5 EU/mL @ 2mg/mL.
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. For corresponding monomers, see Catalog# SPR-520. The unphosphorylated construct is Catalog# SPR-322.

Biological Description

Alternative Names	Alpha synuclein, Alpha-synuclein pre-formed fibril, Alpha synuclein protein PFF, Alpha synuclein PFF, 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
Swiss Prot	P37840
Scientific Background	Serine 129 is the C-terminal serine characteristic to mammalian alpha synuclein, with this serine being determined to be a major phosphorylation site (1). Lewy Bodies in Parkinson’s Disease (PD) and other related synucleinopathies are comprised of alpha synuclein phosphorylated at serine 129 and this phosphorylation may contribute to an increased propensity to aggregate (2). Due to phosphorylation at serine 129 being one of the most abundant PTMs, several studies reported on the PTM as a potential biomarker (3). Our Alpha Synuclein Ser129 Pre-Formed Fibrils are generated in-vitro from purified phosphorylated monomer and phosphorylation is confirmed with our anti-ASYN pS129 monoclonal antibody (Catalog# SMC-600).
References	1. Okochi et al. 2000. Constitutive phosphorylation of the Parkinson’s Disease Associated a-Synuclein. The Journal of Biological Chemistry. DOI: 10.1074/jbc.275.1.390 2. Fujiwara et al. 2002. α-Synuclein is phosphorylated in synucleinopathy lesions. Nature Cell Biology. DOI: 10.1038/ncb748 3. Magalhaes and Lashuel. 2002. Opportunities and challenges of alpha-synuclein as a potential biomarker for Parkinson’s disease and other synucleinopathies. Npj Parkinsons Disease. DOI: 10.1038/s41531-022-00357-0

Product Images

<p>Representative TEM image of Human Recombinant Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521), 200nm scale. Negative stain transmission electron microscopy images of SPR-521 acquired at 80 Kv on carbon coated 400 mesh copper grids using phosphotungstic acid and uranyl acetate stain.</p>

Representative TEM image of Human Recombinant Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521), 200nm scale. Negative stain transmission electron microscopy images of SPR-521 acquired at 80 Kv on carbon coated 400 mesh copper grids using phosphotungstic acid and uranyl acetate stain.

<p>Sedimentation assay on Human Recombinant Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521). Samples were spun down at 15,000 x g, washed, and then spun down again. Fibril samples are prepared in denaturing conditions prior to running on the gel. SDS-PAGE analysis on a 12% Bis-Tris gel shows that the majority of the fibril is insoluble.</p>

Sedimentation assay on Human Recombinant Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521). Samples were spun down at 15,000 x g, washed, and then spun down again. Fibril samples are prepared in denaturing conditions prior to running on the gel. SDS-PAGE analysis on a 12% Bis-Tris gel shows that the majority of the fibril is insoluble.

<p>Dot Blot of purified Human Recombinant Alpha Synuclein pSer129 Monomers (starting material for SPR-521) using Stressmarq’s SMC-600 (anti-ASYN pS129) and SPC-800 (anti-ASYN) confirming phosphorylation in SPR-520, compared to SPR-321. Protein was blotted on nitrocellulose, incubated with 1:1000 primary antibodies and/or 1:4000 secondary antibodies. Secondary control is goat-anti rabbit:HRP. Exposed 1 second.</p>

Dot Blot of purified Human Recombinant Alpha Synuclein pSer129 Monomers (starting material for SPR-521) using Stressmarq’s SMC-600 (anti-ASYN pS129) and SPC-800 (anti-ASYN) confirming phosphorylation in SPR-520, compared to SPR-321. Protein was blotted on nitrocellulose, incubated with 1:1000 primary antibodies and/or 1:4000 secondary antibodies. Secondary control is goat-anti rabbit:HRP. Exposed 1 second.

<p>Modified/total phosphorylation PTM spectrum counts of 4 sites on human alpha synuclein pSer129 monomers (starting material for SPR-521) as determined by mass spectrometry (ScaffoldPTM, localization probability = 100% at both 0% and 95% min. localization). Trypsin/CNBr digestion was used to accurately determine the presence of phosphorylation at and around S129. Note that total counts may include longer peptides where phosphorylation may be more difficult to detect, and most phospho-modified sites that were detected are at S129.</p>

Modified/total phosphorylation PTM spectrum counts of 4 sites on human alpha synuclein pSer129 monomers (starting material for SPR-521) as determined by mass spectrometry (ScaffoldPTM, localization probability = 100% at both 0% and 95% min. localization). Trypsin/CNBr digestion was used to accurately determine the presence of phosphorylation at and around S129. Note that total counts may include longer peptides where phosphorylation may be more difficult to detect, and most phospho-modified sites that were detected are at S129.

<p>In vitro seeding activity of Human Recombinant Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521) in ThT assay. Alpha Synuclein pSer129 Pre-Formed Fibrils seed fibril formation of Alpha Synuclein pSer129 (SPR-520) over 72 hours. Reactions (100uL) shaken at 600 rpm in Greiner-Bio 96 Well Non-Binding Cell Culture Microplates, Black (Greiner-Bio Catalog #655900) at 37oC in the presence of 25 uM ThT and read with an XPS Microplate Reader set at 450nmex/485nmem.</p>

In vitro seeding activity of Human Recombinant Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521) in ThT assay. Alpha Synuclein pSer129 Pre-Formed Fibrils seed fibril formation of Alpha Synuclein pSer129 (SPR-520) over 72 hours. Reactions (100uL) shaken at 600 rpm in Greiner-Bio 96 Well Non-Binding Cell Culture Microplates, Black (Greiner-Bio Catalog #655900) at 37oC in the presence of 25 uM ThT and read with an XPS Microplate Reader set at 450nmex/485nmem.

<p>Representative TEM image of Human Recombinant Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521), 500nm scale. Negative stain transmission electron microscopy images of SPR-521 acquired at 80 Kv on carbon coated 400 mesh copper grids using phosphotungstic acid and uranyl acetate stain.</p>

Representative TEM image of Human Recombinant Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521), 500nm scale. Negative stain transmission electron microscopy images of SPR-521 acquired at 80 Kv on carbon coated 400 mesh copper grids using phosphotungstic acid and uranyl acetate stain.

<p>Atomic force microscopy analysis of Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521) diluted to 0.02 mg/mL with dH2O mounted on freshly cleaved mica, washed, dried and analyzed with tapping mode. Representative images are 5 x 5 µm x-y (left) and 2 x 2 µm x-y (right), both with a Z-range set at 17nm.</p>

Atomic force microscopy analysis of Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521) diluted to 0.02 mg/mL with dH2O mounted on freshly cleaved mica, washed, dried and analyzed with tapping mode. Representative images are 5 x 5 µm x-y (left) and 2 x 2 µm x-y (right), both with a Z-range set at 17nm.

<p>SDS-PAGE analysis of purified Human Recombinant Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521) on a 5-12% gradient Bis-Tris gel. NOTE fibrils are prepared in denaturing conditions before running on the gel.</p>

SDS-PAGE analysis of purified Human Recombinant Alpha Synuclein pSer129 Pre-Formed Fibrils (SPR-521) on a 5-12% gradient Bis-Tris gel. NOTE fibrils are prepared in denaturing conditions before running on the gel.

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