Tau-441 (2N4R) P301S Mutant Monomers (CHO-expressed, N-glycosylated)

Name: Tau-441 (2N4R) P301S Mutant Monomers (CHO-expressed, N-glycosylated)
SKU: SPR-515
Availability: InStock

Human Recombinant Tau-441 (2N4R) P301S Mutant Monomers (CHO-expressed, N-glycosylated)

Catalog No. SPR-515

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Expression System	Chinese Hamster Ovary (CHO)
Tags	N-term histidine tag & TEV site

SKU: SPR-515 Category: Proteins

Mass spectrometry of human P301S Tau 2N4R monomers expressed using CHO cells (SPR-515).

Dot Blot of purified hTau (2N4R) P301S monomers (SPR-515)

Product Name Tau-441 (2N4R) P301S Mutant Monomers (CHO-expressed, N-glycosylated)

Description

Human Recombinant Tau-441 (2N4R) P301S Mutant Monomers (CHO-expressed, N-glycosylated)

Applications WB, SDS PAGE, In vitro Assay

Concentration Lot/batch specific. See included datasheet.

Conjugates

N-term histidine tag & TEV site

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 Chinese Hamster Ovary (CHO)

Amino Acid Sequence GGSHHHHHHHHHHGSGGSENLYFQGMAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVSGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

Purity >95%

Protein Length 441 aa (excluding tag), 466 aa (including tag)

Protein Size 48.609 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 PB 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	Affinity Purified and Size Exclusion
Cite This Product	Human Recombinant Tau-441 (2N4R) P301S Mutant Monomers (CHO-expressed, N-glycosylated) (StressMarq Biosciences Inc., Victoria BC CANADA, Catalog # SPR-515)
Certificate of Analysis	Protein certified >95% pure on SDS-PAGE & Nanodrop analysis
Other Relevant Information	CHO expression in mammalian cell line may lead to more “human” like phosphorylation/glycosylation patterns. For corresponding PFFs, see catalog# SPR-516.

Biological Description

Alternative Names	MAPT, intracellular neurofibrillary tangles, NFTs, paired helical filaments, PHFs, 2N4R
Research Areas	Alzheimer's Disease, Neurodegeneration, Neuroscience, Tangles & Tau
Swiss Prot	P10636-8
Scientific Background	Mammalian N-glycosylation is present on CHO-secreted tau 2N4R, which contributes to slower migration on SDS-PAGE than E.coli or Baculovirus/Sf9 expressed tau (1, 2). N-glycosylated tau has been identified in human AD-diseased brains, but not healthy brains, and may precede tau hyperphosphorylation (3, 4). N-glycosylation of Tau has been demonstrated to affect its aggregation propensity (5). The tau P301S mutation is associated with early onset neurodegeneration, and functionally reduces microtubule assembly and stimulates fibril assembly (6, 7). Our CHO-expressed Tau 2N4R P301S will readily form fibrils in the absence of heparin and contains mammalian post-translational modifications that may better mimic tau in human AD-brains.
References	1. Guo et al., 2019. A pathogenic tau fragment compromises microtubules, disrupts insulin signaling and induces the unfolded protein response. Acta Neuropathologica Communications. DOI: 10.1186/s40478-018-0651-9 2. Losev et al., 2020. Differential effects of putative N-glycosylation sites in human Tau on Alzheimer’s disease-related neurodegeneration. Cellular and Molecular Life Sciences. DOI: 10.1007/s00018-020-03643-3 3. Zhang et al., 2020. Integrative glycoproteomics reveals protein N-glycosylation aberrations and glycoproteomic network alterations in Alzheimer’s disease. Sci. Adv. DOI: 10.1126/sciadv.abc5802 4. Liu et al., 2002. Role of glycosylation in hyperphosphorylation of tau in Alzheimer’s disease. FEBS. DOI: 10.1016/S0014-5793(02)02228-7 5. Losev et al., 2019. Novel model of secreted human tau protein reveals the impact of the abnormal N-glycosylation of tau on its aggregation propensity. Sci. Rep. https://doi.org/10.1038/s41598-019-39218-x 6. Bugiani et al., 1999. Frontotemporal Dementia and Corticobasal Degeneration in a Family with a P301S Mutation in Tau. J Neuropathol Exp Neurol. doi: 10.1097/00005072-199906000-00011. 7. Goedert and Crowther, 1999. Effects of frontotemporal dementia FTDP-17 mutations on heparin-induced assembly of tau filaments. FEBS Lett. DOI: 10.1016/s0014-5793(99)00508-6

Product Images

<p>SDS-PAGE and anti-tau western blot of SPR-515. The majority of CHO-expressed tau 2N4R P301S runs higher (75-100 kDa) than E.coli expressed tau (50-75 kDa) due to post-translational modifications as observed on a 5-12% gradient Bis-Tris gel (left). Tau was confirmed by running on a 12% Tris-Glycine gel, transferring to nitrocellulose, and blotting with 1:1000 anti-tau rabbit polyclonal antibody SPC-801 primary antibody, followed by 1:4000 goat anti-rabbit HRP (right). Exposure time 1 second after 5 minute incubation with chemiluminescent HRP substrate (Moss).</p>

SDS-PAGE and anti-tau western blot of SPR-515. The majority of CHO-expressed tau 2N4R P301S runs higher (75-100 kDa) than E.coli expressed tau (50-75 kDa) due to post-translational modifications as observed on a 5-12% gradient Bis-Tris gel (left). Tau was confirmed by running on a 12% Tris-Glycine gel, transferring to nitrocellulose, and blotting with 1:1000 anti-tau rabbit polyclonal antibody SPC-801 primary antibody, followed by 1:4000 goat anti-rabbit HRP (right). Exposure time 1 second after 5 minute incubation with chemiluminescent HRP substrate (Moss).

<p>PNGase F treatment of SPR-515 shows an observable shift in apparent MW, indicating the presence of N-glycosylation. Monomers were treated with PNGase F (NEB), a glycosidase which specifically cleaves between the innermost GlcNAc and asparagine residues of N-linked oligosaccharides, and incubated at 37oC for 1 hour and run on a 5-12% gradient Bis-Tris gel.</p>

PNGase F treatment of SPR-515 shows an observable shift in apparent MW, indicating the presence of N-glycosylation. Monomers were treated with PNGase F (NEB), a glycosidase which specifically cleaves between the innermost GlcNAc and asparagine residues of N-linked oligosaccharides, and incubated at 37oC for 1 hour and run on a 5-12% gradient Bis-Tris gel.

<p>Modified/Total deamidation spectrum counts as determined by mass spectrometry of SPR-515 before and after PNGase F treatment identifies potential N-glycosylation sites at N167, N359 and N410. Blue color indicates deamidation sites that match the N-glycosylation motif (N-X-S/T/C) and have a higher deamidation count after PNGase F treatment. No deamidation was present at N167 or N410 without PNGase F, suggesting these residues are protected from nonspecific deamidation by N-glycosylation. Some deamidation was present at N359 without PNGase F treatment, indicating a population of monomers is not glycosylated at this position. Several non-consensus, non-PNGase F-dependent deamidation sites were present, which may have occurred during production or the mass spectrometry workflow. Both CID and HCD fragmentation methods were used to improve sequence coverage and deamidation detection. Overall protein sequence coverage was 82%, with a localization probability cutoff set at ≥95%.</p>

Modified/Total deamidation spectrum counts as determined by mass spectrometry of SPR-515 before and after PNGase F treatment identifies potential N-glycosylation sites at N167, N359 and N410. Blue color indicates deamidation sites that match the N-glycosylation motif (N-X-S/T/C) and have a higher deamidation count after PNGase F treatment. No deamidation was present at N167 or N410 without PNGase F, suggesting these residues are protected from nonspecific deamidation by N-glycosylation. Some deamidation was present at N359 without PNGase F treatment, indicating a population of monomers is not glycosylated at this position. Several non-consensus, non-PNGase F-dependent deamidation sites were present, which may have occurred during production or the mass spectrometry workflow. Both CID and HCD fragmentation methods were used to improve sequence coverage and deamidation detection. Overall protein sequence coverage was 82%, with a localization probability cutoff set at ≥95%.

<p>Modified/Total phosphorylation PTM spectrum counts reveal up to 7 phosphorylation sites on human P301S Tau 2N4R monomers expressed using CHO as determined by mass spectrometry. Both CID and HCD fragmentation methods were used to improve sequence coverage and deamidation detection. Protein sequence coverage was 82%. Localization probability cutoff set at ≥80% (yellow) or ≥95% (green). Note: number of phosphorylation sites appear less than Baculovirus/Sf9 expressed tau 2N4R (see StressMarq cat# SPR-471, 472, 496 and 498).</p>

Modified/Total phosphorylation PTM spectrum counts reveal up to 7 phosphorylation sites on human P301S Tau 2N4R monomers expressed using CHO as determined by mass spectrometry. Both CID and HCD fragmentation methods were used to improve sequence coverage and deamidation detection. Protein sequence coverage was 82%. Localization probability cutoff set at ≥80% (yellow) or ≥95% (green). Note: number of phosphorylation sites appear less than Baculovirus/Sf9 expressed tau 2N4R (see StressMarq cat# SPR-471, 472, 496 and 498).

<p>Dot Blot of purified hTau (2N4R) P301S monomers (SPR-515) using Stressmarq’s SPC-801 and a phospho-S324 Tau antibody (GeneBio Systems) comparing phosphorylation in E.coli-expressed, baculovirus/sf9-expressed, and CHO-expressed material. 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 hTau (2N4R) P301S monomers (SPR-515) using Stressmarq’s SPC-801 and a phospho-S324 Tau antibody (GeneBio Systems) comparing phosphorylation in E.coli-expressed, baculovirus/sf9-expressed, and CHO-expressed material. 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.

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