Human Notch-1 Intracellular Domain Antibody
R&D Systems, part of Bio-Techne | Catalog # AF3647
Key Product Details
Validated by
Biological Validation
Species Reactivity
Validated:
Human
Cited:
Human
Applications
Validated:
Chromatin Immunoprecipitation (ChIP), CyTOF-ready, Flow Cytometry, Immunocytochemistry, Western Blot
Cited:
Flow Cytometry, Immunocytochemistry, Immunohistochemistry, Western Blot
Label
Unconjugated
Antibody Source
Polyclonal Sheep IgG
Product Specifications
Immunogen
E. coli-derived recombinant human Notch-1
Gly2428-Lys2556
Accession # P46531
Gly2428-Lys2556
Accession # P46531
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.
Clonality
Polyclonal
Host
Sheep
Isotype
IgG
Scientific Data Images for Human Notch-1 Intracellular Domain Antibody
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.
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.
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. Thec-mycpromoter was detected by standard PCR.Applications for Human Notch-1 Intracellular Domain Antibody
Application
Recommended Usage
Chromatin Immunoprecipitation (ChIP)
5 µg/5 x 106 cells
Sample: PMA and calcium ionomycin treated Jurkat human acute T cell leukemia cell line chromatin, c-myc promoter detected by standard PCR
Sample: PMA and calcium ionomycin treated Jurkat human acute T cell leukemia cell line chromatin, c-myc promoter detected by standard PCR
CyTOF-ready
Ready to be labeled using established conjugation methods. No BSA or other carrier proteins that could interfere with conjugation.
Flow Cytometry
2.5 µg/106 cells
Sample: U2OS human osteosarcoma cell line
Sample: U2OS human osteosarcoma cell line
Immunocytochemistry
5-15 µg/mL
Sample: Immersion fixed Saos-2 human osteosarcoma cell line
Sample: Immersion fixed Saos-2 human osteosarcoma cell line
Western Blot
1 µg/mL
Sample: 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, transfected with full length human Notch-1
Sample: 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, transfected with full length human Notch-1
Please Note: Optimal dilutions of this antibody should be experimentally determined.
Reviewed Applications
Read 4 reviews rated 3.8 using AF3647 in the following applications:
Formulation, Preparation, and Storage
Purification
Antigen Affinity-purified
Reconstitution
Reconstitute at 0.2 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.
Formulation
Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
Shipping
Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.
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.
Alternate Names
Notch1, TAN1
Gene Symbol
NOTCH1
UniProt
Additional Notch-1 Products
Product Documents for Human Notch-1 Intracellular Domain Antibody
Product Specific Notices for Human Notch-1 Intracellular Domain Antibody
For research use only
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