Human Notch-2 Intracellular Domain Antibody

R&D Systems | Catalog # AF3735

R&D Systems

Key Product Details

Species Reactivity

Validated:

Human

Cited:

Human

Applications

Validated:

Immunohistochemistry, Western Blot

Cited:

Immunohistochemistry-Frozen, Western Blot, Flow Cytometry

Label

Unconjugated

Antibody Source

Polyclonal Goat IgG
View all Notch-2 Primary Antibodies »

Product Specifications

Immunogen

E. coli-derived recombinant human Notch‑2
Asp2063-Pro2413
Accession # Q04721

Specificity

Detects human Notch-2 Intracellular Domain in direct ELISAs and Western blots. In direct ELISAs and Western blots, approximately 5% cross‑reactivity with recombinant human (rh) Notch-3 (aa 2195‑2321), rhNotch-6 (aa 2251‑2556) and rhNotch-4 (aa 1778‑2003) is observed.

Clonality

Polyclonal

Host

Goat

Isotype

IgG

Scientific Data Images for Human Notch-2 Intracellular Domain Antibody

Detection of Notch‑2 in Human Kidney.

Notch‑2 was detected in immersion fixed paraffin-embedded sections of human kidney using Goat Anti-Human Notch‑2 Intracellular Domain Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3735) at 5 µg/ml for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC004) or the HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (Catalog # VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to the cytoplasm and nucleus. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.
Detection of Notch-2 Intracellular Domain by Western Blot

Detection of Notch-2 Intracellular Domain by Western Blot

Interaction of Notch2 and Jagged1 between CAFs and lung cancer cells. (A) Expression of Notches and their ligands in lung cancer cells and their CAFs, respectively. (B) Close interaction of Nocth2 and Jagged1 between H1299 cells and CAFs as determined by proximity ligation assay (PLA). H1299-GFP cells, CAFs (BioTracker 400 Blue cytoplasmic membrane dye), and H1299-GFP cells mixed with CAFs were committed to PLA analysis (red). (C) Increased NOTCH 2 intracellular domain (N2ICD) nuclear translocation in H1299 cells. Cells were collected and the nuclear and cytosol fractions were extracted using a commercial kit. Equal levels of protein were assessed by immunoblotting. (D) Inhibition of Jagged1 in CAFs reduced the VM formation of H1299 cells. Knockdown of Jagged 1 in CAFs reduced (E) N2ICD nuclear translocation, and (F) HEY1 upregulation driven by CAFs in H1299 cells. Knockdown of Notch2 in H1299 reduced (G) VM formation, (H) N2ICD nuclear translocation, and (I) HEY1 upregulation driven by CAFs. CAFs and H1299 cells were transfected with a control, Jagged1 (for CAFs), or Notch2 (for H1299 cells) siRNA for 24 h, then CAFs were mixed with H1299 cells. The HEY1 mRNA and nuclear N2ICD levels were detected using a quantitative reverse transcription polymerase chain reaction and immunoblotting, respectively. All results are representative of at least three independent experiments. Each value is the mean ± SD of three determinations; *P < 0.05. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34485133), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Notch-2 Intracellular Domain by Western Blot

Detection of Notch-2 Intracellular Domain by Western Blot

Interaction of Notch2 and Jagged1 between CAFs and lung cancer cells. (A) Expression of Notches and their ligands in lung cancer cells and their CAFs, respectively. (B) Close interaction of Nocth2 and Jagged1 between H1299 cells and CAFs as determined by proximity ligation assay (PLA). H1299-GFP cells, CAFs (BioTracker 400 Blue cytoplasmic membrane dye), and H1299-GFP cells mixed with CAFs were committed to PLA analysis (red). (C) Increased NOTCH 2 intracellular domain (N2ICD) nuclear translocation in H1299 cells. Cells were collected and the nuclear and cytosol fractions were extracted using a commercial kit. Equal levels of protein were assessed by immunoblotting. (D) Inhibition of Jagged1 in CAFs reduced the VM formation of H1299 cells. Knockdown of Jagged 1 in CAFs reduced (E) N2ICD nuclear translocation, and (F) HEY1 upregulation driven by CAFs in H1299 cells. Knockdown of Notch2 in H1299 reduced (G) VM formation, (H) N2ICD nuclear translocation, and (I) HEY1 upregulation driven by CAFs. CAFs and H1299 cells were transfected with a control, Jagged1 (for CAFs), or Notch2 (for H1299 cells) siRNA for 24 h, then CAFs were mixed with H1299 cells. The HEY1 mRNA and nuclear N2ICD levels were detected using a quantitative reverse transcription polymerase chain reaction and immunoblotting, respectively. All results are representative of at least three independent experiments. Each value is the mean ± SD of three determinations; *P < 0.05. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34485133), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Notch-2 Intracellular Domain by Western Blot

Detection of Notch-2 Intracellular Domain by Western Blot

Interaction of Notch2 and Jagged1 between CAFs and lung cancer cells. (A) Expression of Notches and their ligands in lung cancer cells and their CAFs, respectively. (B) Close interaction of Nocth2 and Jagged1 between H1299 cells and CAFs as determined by proximity ligation assay (PLA). H1299-GFP cells, CAFs (BioTracker 400 Blue cytoplasmic membrane dye), and H1299-GFP cells mixed with CAFs were committed to PLA analysis (red). (C) Increased NOTCH 2 intracellular domain (N2ICD) nuclear translocation in H1299 cells. Cells were collected and the nuclear and cytosol fractions were extracted using a commercial kit. Equal levels of protein were assessed by immunoblotting. (D) Inhibition of Jagged1 in CAFs reduced the VM formation of H1299 cells. Knockdown of Jagged 1 in CAFs reduced (E) N2ICD nuclear translocation, and (F) HEY1 upregulation driven by CAFs in H1299 cells. Knockdown of Notch2 in H1299 reduced (G) VM formation, (H) N2ICD nuclear translocation, and (I) HEY1 upregulation driven by CAFs. CAFs and H1299 cells were transfected with a control, Jagged1 (for CAFs), or Notch2 (for H1299 cells) siRNA for 24 h, then CAFs were mixed with H1299 cells. The HEY1 mRNA and nuclear N2ICD levels were detected using a quantitative reverse transcription polymerase chain reaction and immunoblotting, respectively. All results are representative of at least three independent experiments. Each value is the mean ± SD of three determinations; *P < 0.05. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34485133), licensed under a CC-BY license. Not internally tested by R&D Systems.

Applications for Human Notch-2 Intracellular Domain Antibody

Application
Recommended Usage

Immunohistochemistry

5-15 µg/mL
Sample: Immersion fixed paraffin-embedded sections of human kidney

Western Blot

0.1 µg/mL
Sample: Recombinant Human Notch‑2 Intracellular Domain

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. See Certificate of Analysis for details.<br/>*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.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Notch-2

Human Notch-2 is a 300 kDa type I transmembrane glycoprotein that is one of four human Notch homologues involved in developmental processes (1‑3). Although Notch proteins are structurally and functionally similar, deletion of either Notch-1 or Notch-2 is lethal, showing that not all functions overlap (4, 5). Mice with hypomorphic Notch-2 show defects in the development of kidney, heart and eyes (6). Notch-2 is up‑regulated in mature B cells and is critical for differentiation to splenic marginal zone B cells (7). Notch-2 is also preferentially expressed in choroid plexus epithelia and neuronal precursors (8, 9). Human Notch-2 is synthesized as a 2471 amino acid (aa) precursor that contains a 25 aa signal sequence, a 1652 aa extracellular domain (ECD), and a 794 aa transmembrane (TM) and cytoplasmic segment. The ECD contains 35 EGF-like repeats and three Lin-12/Notch repeats, while the cytoplasmic region shows six ankyrin repeats, a glutamine-rich domain and a PEST sequence. Binding of ligands, including Jagged and Delta-like molecules in humans, has been localized to the 11th and 12th EGF-like repeats of Notch (10). Notch receptors undergo post-translational furin-type proteolytic cleavage (11). This forms a heterodimer through the interaction of a hydrophobic area in the ligand‑binding extracellular region with the TM/cytoplasmic portion (11, 12). Upon ligand binding, additional sequential proteolysis by TNF-converting enzyme (ADAM‑17) and the presenilin-dependent gamma -secretase results in the release of the Notch intracellular domain (NICD) which translocates into the nucleus, activating transcription of Notch-responsive genes (13). Human Notch-2 ECD (aa 26‑530) shows 93%, 93%, 96% and 96% aa identity with the corresponding regions of mouse, rat, canine, and bovine Notch-2, respectively. This region also exhibits approximately 60% aa identity with human Notch-1 and Notch-3.

References

  1. Stifani, S. et al. (1992) Nat. Genet. 2:119.
  2. Dumortier, A. et al. (2005) Int. J. Hematol. 82:277.
  3. Yoon, K. and N. Gaiano (2005) Nat. Neurosci. 8:709.
  4. Hamada, Y. et al. (1999) Development 126:3415.
  5. Shimizu, K. et al. (2002) Biochem. Biophys. Res. Comm. 291:775.
  6. McCright, B. et al. (2001) Development 128:491.
  7. Saito, T. et al. (2003) Immunity 18:675.
  8. Irvin, D. K. et al. (2001) J. Comp. Neurol. 436:167.
  9. Solecki, D. J. et al. (2001) Neuron 31:557.
  10. Hambleton, S. et al. (2004) Structure 12:2173.
  11. Logeat, F. et al. (1998) Proc. Natl. Acad. Sci. USA 95:8108.
  12. Sanchez-Irizarry, C. et al. (2004) Mol. Cell. Biol. 24:9265.
  13. Mumm, J.S. and R. Kopan (2000) Dev. Biol. 228:151.

Alternate Names

Notch2

Entrez Gene IDs

4853 (Human); 29492 (Rat)

Gene Symbol

NOTCH2

UniProt

Q04721

Additional Notch-2 Products

Product Documents for Human Notch-2 Intracellular Domain Antibody

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Product Specific Notices for Human Notch-2 Intracellular Domain Antibody

For research use only

Citations for Human Notch-2 Intracellular Domain Antibody

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