AF3647

antibody from R&D Systems

Targeting: NOTCH1 TAN1

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Flow cytometry (Data presented on provider website)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Antibody Data

Product number

AF3647

Provider

R&D Systems

Product name

Human Notch-1 Intracellular Domain Antibody

Antibody type

Polyclonal

Description

Immunogen affinity purified. 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.

Reactivity

Human

Host

Sheep

Conjugate

Unconjugated

Antigen sequence

P46531


Isotype

IgG

Vial size

100 ug

Concentration

LYOPH

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.

References

Submitted references

NOTCH1-Dependent Nitric Oxide Signaling Deficiency in Hypoplastic Left Heart Syndrome Revealed Through Patient-Specific Phenotypes Detected in Bioengineered Cardiogenesis.

Hrstka SC, Li X, Nelson TJ, Wanek Program Genetics Pipeline Group.
Stem cells (Dayton, Ohio) 2017 Apr;35(4):1106-1119

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A dual molecular analogue tuner for dissecting protein function in mammalian cells.

Brosh R, Hrynyk I, Shen J, Waghray A, Zheng N, Lemischka IR
Nature communications 2016 May 27;7:11742

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Involvement of Notch in activation and effector functions of γδ T cells.

Gogoi D, Dar AA, Chiplunkar SV
Journal of immunology (Baltimore, Md. : 1950) 2014 Mar 1;192(5):2054-62

Western blot

Supportive validation

Submitted by

R&D Systems (provider)

Main image

Experimental details

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.

Immunocytochemistry

Supportive validation

Submitted by

R&D Systems (provider)

Main image

Experimental details

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.

Chromatin Immunoprecipitation

Supportive validation

Submitted by

R&D Systems (provider)

Main image

Experimental details

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.