Alpha Synuclein E114C Mutant Pre-formed Fibrils: ATTO 488

Name: Alpha Synuclein E114C Mutant Pre-formed Fibrils: ATTO 488
SKU: SPR-518-A488
Availability: InStock

Human Recombinant Alpha Synuclein E114C Mutant Pre-formed Fibrils: ATTO 488

Catalog No. SPR-518-A488

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Expression System	E. coli
Tags	ATTO 488

SKU: SPR-518-A488 Category: Proteins

Representative TEM image of ATTO-488 conjugated Alpha Synuclein E114C Pre-Formed Fibrils (SPR-518-A88)

Fluorescent signal of alpha-synuclein E114C-ATTO-488 PFFs (SPR-518-A88)

In vitro seeding activity of human Alpha Synuclein wildtype monomer in ThT assay. (SPR-518-A88)

Sedimentation assay on ATTO-488 conjugated Alpha Synuclein E114C Pre-Formed Fibrils. (SPR-518-A88)

SDS-PAGE analysis of ATTO-488 conjugated Alpha Synuclein E114C Pre-Formed Fibrils (SPR-518-A88)

Product Name Alpha Synuclein E114C Mutant Pre-formed Fibrils: ATTO 488

Description

Human Recombinant Alpha Synuclein E114C Mutant Pre-formed Fibrils: ATTO 488

Applications WB, Native PAGE, In vitro Assay, In vivo Assay

Concentration Lot/batch specific. See included datasheet.

Conjugates

ATTO 488

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 10% of mixture (mutant conjugated form): MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILCDMPVDPDNEAYEMPSEEGYQDYEPEA
90% of mixture (wildtype):
MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA

Purity >95%

Protein Length 140 AA

Protein Size 14.434 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	1X 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 & SEC purified
Cite This Product	Human Recombinant Alpha Synuclein E114C Mutant Pre-formed Fibrils: ATTO 488 (StressMarq Biosciences Inc., Victoria BC CANADA, Catalog # SPR-518)
Certificate of Analysis	Protein certified >95% pure on SDS-page and nanodrop analysis, endotoxin below 5 EU/mL at 2 mg/mL on starting monomer material.
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.

Biological Description

Alternative Names	Alpha synuclein pre-formed fibril, Alpha-synuclein PFF, Alpha synuclein protein fibrils, 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's 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	The alpha-synuclein (aSyn) E114C mutation facilitates a single site-specific conjugation with ATTO-488 maleimide that avoids any hindrance on fibrilization or cell entry that may be conferred by non-specific lysine targeting conjugations. This conjugation is ideal due to internal position relative to C-terminal truncation sites, proximity to the NAC, and lack of interference with recruitment in vitro or in primary neurons (1, 2). Pre-formed fibrils (PFFs) generated with 5-25% fluorescently tagged E114C mutants have demonstrated a relative potency >80% compared to wild-type aSyn for inducing misfolding of endogenous aSyn, indicating no significant perturbation of seeding in living cells (1). Atto-488 is a useful tool for identifying cell entry, as the addition of Trypan Blue to cultures prior to imaging will quench fluorescence of extracellular Atto-488 conjugated aSyn (3). Our aSyn E114C-Atto-488 PFFs, which are formed from 10% fluorescently tagged E114C mutants and 90% wild-type monomers, are an excellent tool for studying cell entry and localization, with demonstrated entry into neurons after trypan blue quenching.
References	1.,Haney et al. 2016. Comparison of strategies for non-perturbing labeling of α-synuclein to study amyloidogenesis. Organic & Biomolecular Chemistry. DOI: 10.1039/c5ob02329g 2.,Karpowicz et al. 2017. Selective imaging of internalized proteopathic a-synuclein seeds in primary neurons reveals mechanistic insight into transmission of synucleinopathies. JBC. DOI: 10.1074/jbc.M117.780296 3.,Pieri et al. 2016. Structural and functional properties of prefibrillar α-synuclein oligomers. Scientific Reports. DOI: 10.1038/srep24526

Product Images

<p>Neuronal uptake of ATTO-488 conjugated Alpha Synuclein E114C Pre-Formed Fibril (PFFs) (SPR-518-A88) visible by fluorescence after Trypan Blue quenching. Neurons expressing mCherry via AAVs (division 19) were treated with SPR-518 and then Trypan Blue to quench extracellular PFF fluorescence. ATTO-488 fluorescence present after (bottom row) Trypan Blue addition is from internalized PFFs. Note: greater mCherry signal post-Trypan Blue addition attributed to overlap of excitation/emission spectra (mCherry maxima ex 587/em610, Trypan Blue ex maxima 560/em630).</p>

Neuronal uptake of ATTO-488 conjugated Alpha Synuclein E114C Pre-Formed Fibril (PFFs) (SPR-518-A88) visible by fluorescence after Trypan Blue quenching. Neurons expressing mCherry via AAVs (division 19) were treated with SPR-518 and then Trypan Blue to quench extracellular PFF fluorescence. ATTO-488 fluorescence present after (bottom row) Trypan Blue addition is from internalized PFFs. Note: greater mCherry signal post-Trypan Blue addition attributed to overlap of excitation/emission spectra (mCherry maxima ex 587/em610, Trypan Blue ex maxima 560/em630).

<p>Representative TEM image of ATTO-488 conjugated Alpha Synuclein E114C Pre-Formed Fibrils (SPR-518-A88), 500nm scale. Negative stain transmission electron microscopy images of SPR-518 acquired at 80 Kv on carbon coated 400 mesh copper grids using phosphotungstic acid and uranyl acetate stain.</p>

Representative TEM image of ATTO-488 conjugated Alpha Synuclein E114C Pre-Formed Fibrils (SPR-518-A88), 500nm scale. Negative stain transmission electron microscopy images of SPR-518 acquired at 80 Kv on carbon coated 400 mesh copper grids using phosphotungstic acid and uranyl acetate stain.

<p>Representative TEM image of ATTO-488 conjugated Alpha Synuclein E114C Pre-Formed Fibrils (SPR-518-A88), 200nm scale. Negative stain transmission electron microscopy images of SPR-518 acquired at 80 Kv on carbon coated 400 mesh copper grids using phosphotungstic acid and uranyl acetate stain.</p>

Representative TEM image of ATTO-488 conjugated Alpha Synuclein E114C Pre-Formed Fibrils (SPR-518-A88), 200nm scale. Negative stain transmission electron microscopy images of SPR-518 acquired at 80 Kv on carbon coated 400 mesh copper grids using phosphotungstic acid and uranyl acetate stain.

<p>Fluorescent signal of alpha-synuclein E114C-ATTO-488 PFFs (SPR-518-A88). SPR-518 concentration and fluorescence (excitation 488nm, emission 521 nm) exhibit a linear relationship at all concentrations tested (250 ng/mL – 500 µg/mL).</p>

Fluorescent signal of alpha-synuclein E114C-ATTO-488 PFFs (SPR-518-A88). SPR-518 concentration and fluorescence (excitation 488nm, emission 521 nm) exhibit a linear relationship at all concentrations tested (250 ng/mL – 500 µg/mL).

<p>In vitro seeding activity of human Alpha Synuclein wildtype monomer in ThT assay (SPR-518-A88). SPR-518 (ATTO-488 conjugated Alpha Synuclein E114C fibrils) seed fibril formation of SPR-321 (Type 1 Alpha Synuclein wildtype monomer) 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 and read with an XPS Microplate Reader set at 450nmex/485nmem.</p>

In vitro seeding activity of human Alpha Synuclein wildtype monomer in ThT assay (SPR-518-A88). SPR-518 (ATTO-488 conjugated Alpha Synuclein E114C fibrils) seed fibril formation of SPR-321 (Type 1 Alpha Synuclein wildtype monomer) 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 and read with an XPS Microplate Reader set at 450nmex/485nmem.

<p>Sedimentation assay on ATTO-488 conjugated Alpha Synuclein E114C Pre-Formed Fibrils (SPR-518-A88). Samples were pelleted at 15,000 x g, washed, and then re-centrifuged. 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 fibril is insoluble.</p>

Sedimentation assay on ATTO-488 conjugated Alpha Synuclein E114C Pre-Formed Fibrils (SPR-518-A88). Samples were pelleted at 15,000 x g, washed, and then re-centrifuged. 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 fibril is insoluble.

<p>SDS-PAGE analysis of ATTO-488 conjugated Alpha Synuclein E114C Pre-Formed Fibrils (SPR-518-A88) on a 12% Tris-Glycine gel. Fibril samples are prepared in denaturing conditions prior to running on the gel. Note: PFFs are not SDS-stable, consistent with wild-type PFFs.</p>

SDS-PAGE analysis of ATTO-488 conjugated Alpha Synuclein E114C Pre-Formed Fibrils (SPR-518-A88) on a 12% Tris-Glycine gel. Fibril samples are prepared in denaturing conditions prior to running on the gel. Note: PFFs are not SDS-stable, consistent with wild-type PFFs.

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