Human Notch-1 Intracellular Domain Antibody

(3 citations)
(2 Reviews)
  
  • Species Reactivity
    Human
  • Specificity
    Detects the intracellular domain (ICD) of human Notch-1 in direct ELISAs and Western blots. In direct ELISAs and Western blots,  less than 1% cross-reactivity with recombinant human (rh) Notch-2 ICD, rhNotch-3 ICD, and rhNotch-4 ICD is observed.
  • Source
    Polyclonal Sheep IgG
  • Purification
    Antigen Affinity-purified
  • Immunogen
    E. coli-derived recombinant human Notch‑1
    Gly2428-Lys2556
    Accession # P46531
  • Formulation
    Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied as a 0.2 µm filtered solution in PBS.
  • Label
    Unconjugated
Applications
  •  
    Recommended
    Concentration
    Sample
  • Western Blot
    1 µg/mL
    See below
  • Flow Cytometry
    2.5 µg/106 cells
    U2OS human osteosarcoma cell line
  • Chromatin Immunoprecipitation (ChIP)
    5 µg/5 x 106 cells
    See below
  • CyTOF-ready
    Ready to be labeled using established conjugation methods. No BSA or other carrier proteins that could interfere with conjugation.
  • Immunocytochemistry
    5-15 µg/mL
    See below
Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website.
Data Examples
Detection of Human Notch-1.
Western blot shows lysates of MOLT‑4 human acute lymphoblastic leukemia cell line, Jurkat human acute T cell leukemia cell line, RPMI 8226 human multiple myeloma cell line, 293T human embryonic kidney cell line, and 293T human embryonic kidney cell line (1 µg per lane), transfected with full length human Notch‑1. PVDF membrane was probed with 1 µg/mL of Sheep Anti-Human Notch‑1 Intracellular Domain Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3647) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). Specific bands were detected for Notch-1 intracellular domain (NICD) and full length Notch‑1 (Notch‑1 FL) at approximately 115 and 300 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
Chromatin Immunoprecipitation (ChIP)
Detection of Notch‑1-regulated Genes by Chromatin Immunoprecipitation. Jurkat human acute T cell leukemia cell line treated with 50 ng/mL PMA and 200 ng/mL calcium ionomycin for 30 minutes was fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. Notch‑1/DNA complexes were immunoprecipitated using 5 μg Sheep Anti-Human Notch‑1 Intracellular Domain Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3647) or control antibody (Catalog # 5-001-A) for 15 minutes in an ultrasonic bath, followed by Biotinylated Anti-Sheep IgG Secondary Antibody (Catalog # BAF016). Immunocomplexes were captured using 50 μL of MagCellect Streptavidin Ferrofluid (Catalog # MAG999) and DNA was purified using chelating resin solution. The c-myc promoter was detected by standard PCR.
Immunocytochemistry
Notch‑1 in Saos-2 Human Cell Line. Notch‑1 was detected in immersion fixed Saos-2 human osteosarcoma cell line using 10 µg/mL Sheep Anti-Human Notch‑1 Intracellular Domain Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3647) for 3 hours at room temperature. Cells were stained with the NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody (red; Catalog # NL010) and counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.
Preparation and Storage
  • Reconstitution
    Reconstitute at 0.2 mg/mL in sterile PBS.
  • Shipping
    The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. *Small pack size (SP) is shipped with polar packs. Upon receipt, store it immediately at -20 to -70 °C
  • Stability & Storage
    Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
    • 12 months from date of receipt, -20 to -70 °C as supplied.
    • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
    • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
Background: Notch-1

Notch-1 (so named for "notches" in fly wings; also TAN-1) is a 300 kDa member of the Notch family of glycoproteins. It is associated with gene activation in both embryo and adult. Human Notch-1 is a 2538 amino acid (aa) type I transmembrane glycoprotein. It undergoes Golgi processing to generate a heterodimer composed of a 180‑200 kDa disulfide-linked extracellular domain (aa 18‑1664) and a 120 kDa membrane-bound segment (aa 1665‑2556). Upon ligand binding, the 110 kDa segment undergoes two cleavages which generate an NICD (notch intracellular domain) (aa 1754‑2556), a nuclear transcription factor. One isoform shows a deletion of aa 248‑288. Over aa 2428‑2556, human Notch 1 is 83% and 89% aa identical to canine and mouse Notch-1, respectively.

  • Entrez Gene IDs:
    4851 (Human); 25496 (Rat)
  • Alternate Names:
    EC 2.1.2.11; EC 3.4.21.68; hN1; Notch (Drosophila) homolog 1 (translocation-associated); notch 1Notch homolog 1, translocation-associated (Drosophila); Notch homolog 1, translocation-associated; Notch1; Notch-1; TAN1; TAN1neurogenic locus notch homolog protein 1; Translocation-associated notch protein TAN-1
Related Research Areas
Citations:

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

3 Citations: Showing 1 - 3
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Species
Applications
Sample Type
  1. NOTCH1-dependent nitric oxide signaling deficiency in hypoplastic left heart syndrome revealed through patient-specific phenotypes detected in bioengineered cardiogenesis
    Authors: SC Hrstka, X Li, TJ Nelson,
    Stem Cells, 2017;0(0):.
    Species: Human
    Sample Type: Cell Lysates
    Application: WB
  2. A dual molecular analogue tuner for dissecting protein function in mammalian cells
    Nat Commun, 2016;7(0):11742.
    Species: Human
    Sample Type: Cell Lysates
    Application: WB
  3. Involvement of Notch in activation and effector functions of gammadelta T cells.
    Authors: Gogoi D, Dar A, Chiplunkar S
    J Immunol, 2014;192(5):2054-62.
Cell and Tissue Staining Kits
Description Application Cat# Citations Images  

Anti-Sheep HRP-DAB Cell & Tissue Staining Kit

CTS019 1
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Isotype Controls
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Normal Sheep IgG Control

Ctrl 5-001-A 9
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Secondary Antibodies
Description Application Cat# Citations Images  

Sheep IgG HRP-conjugated Antibody

WB, Simple Western HAF016 14  
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Donkey Anti-Sheep IgG NorthernLights™ NL557-conjugated Antibody

Flow, IHC, ICC NL010 3
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Donkey Anti-Sheep IgG NorthernLights™ NL637-conjugated Antibody

Flow, IHC, ICC NL011 5
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Donkey Anti-Sheep IgG NorthernLights™ NL493-conjugated Antibody

Flow, IHC, ICC NL012 2
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Sheep IgG (H+L) PE-conjugated Antibody

Flow F0126  
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Donkey Anti-Sheep IgG Biotinylated Antibody

WB BAF016 2
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Sheep IgG (H+L) APC-conjugated Antibody

Flow F0127  
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Sheep IgG VisUCyte HRP Polymer Antibody

IHC VC006  
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Donkey Anti-Sheep IgG (H+L) Fluorescein-conjugated Antibody

Flow F0125 1
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Donkey Anti-Sheep IgG (H+L) PerCP-conjugated Antibody

Flow F0128
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Average Rating: 3.5 (Based on 2 reviews)

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We have 2 reviews tested in 1 species: Human.
We have 2 reviews tested in 2 applications: Immunocytochemistry/Immunofluorescence, Western Blot.

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Images Ratings Applications Species Reviewed By Date Details
Immunocytochemistry/Immunofluorescence Human Notch-1 Intracellular Domain Antibody AF3647
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Average
 ICC/IF Human Anonymous 12/10/2017
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Image Details
Immunocytochemistry/Immunofluorescence Human Notch-1 Intracellular Domain Antibody AF3647
Immunocytochemistry/Immunofluorescence: Human Notch-1 Intracellular Domain Antibody [AF3647]

Summary

ApplicationImmunocytochemistry/Immunofluorescence
Sample TestedBladder tissue,Bladder cancer tissue
SpeciesHuman
Western Blot Human Notch-1 Intracellular Domain Antibody AF3647
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Very Good
 WB Human Anonymous 03/01/2017
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Western Blot Human Notch-1 Intracellular Domain Antibody AF3647
Western Blot: Human Notch-1 Intracellular Domain Antibody [AF3647]

Summary

ApplicationWestern Blot
Sample TestedBreast cancer cells
SpeciesHuman

Other Experimental Details

Other Experimental DetailsExpression of cleaved Notch-1 (Intracellular Domain) in mammary tumor cell lines. Dilution: 1:200 in PBS with 5% BSA. Secondary Ab: anti-Sheep IgG 1:5,000.

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