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

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:

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SPR-515_Tau-441-2N4R-P301S-Mutant-Monomers-CHO-expressed-N-glycosylated-Protein-SDS-Page-1.png
SDS-PAGE of SPR-515 glycosylation.Mass spectrometry of human P301S Tau 2N4R monomers expressed using CHO cells (SPR-515).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 2 mg/ml or 5 mg/ml
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:

λex = 493 nm

λem = 518 nm

εmax = 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:

λex = 652 nm

λem = 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:

λex = 353 nm

λem = 432 nm

εmax = 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:

λex = 400 nm

λem = 420 nm

εmax = 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:

λex = 593 nm

λem = 618 nm

εmax = 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:

λex = 638 nm

λem = 658 nm

εmax = 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:

λex = 482 nm

λem = 677 nm

εmax = 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:

λex = 535 nm

λem = 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:

λex = 494 nm

λem = 520 nm

εmax = 7.3×104

Φf = 0.92

τfl = 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:

λex = 700 nm

λem = 719 nm

εmax = 1.25×105

Φf = 0.25

τfl = 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:

λex = 680 nm

λem = 700 nm

εmax = 1.25×105

Φf = 0.30

τfl = 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:

λex = 663 nm

λem = 684 nm

εmax = 1.25×105

Φf = 0.30

τfl = 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:

λex = 629 nm

λem = 657 nm

εmax = 1.3×105

Φf = 0.64

τfl = 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:

λex = 601 nm

λem = 627 nm

εmax = 1.2×105

Φf = 0.85

τfl = 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:

λex = 563 nm

λem = 592 nm

εmax = 1.2×105

Φf = 0.9

τfl = 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:

λex = 501 nm

λem = 523 nm

εmax = 9.0×104

Φf = 0.80

τfl = 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:

λex = 390 nm

λem = 479 nm

εmax = 2.4×104

Φf = 0.90

τfl = 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:

λex = 650 nm

λem = 660 nm

εmax = 7.0×105

Φf = 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

BiotinBiotin Conjugate Structure

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:

λex = 565 nm

λem = 575 nm

εmax = 2.0×106

Φf = 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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