Notch Activated Targets Antibody Sampler Kit #68309
Product Information
Kit Usage Information
Protocols
- 2936: Western Blotting, Immunohistochemistry (Paraffin)
- 2947: Western Blotting, Immunoprecipitation (Agarose), Immunohistochemistry (Paraffin), Immunofluorescence, Flow
- 3608: Western Blotting, Immunoprecipitation (Agarose), Immunohistochemistry (Paraffin), ChIP Magnetic
- 4147: Western Blotting, Immunoprecipitation (Magnetic), ChIP Magnetic
- 5313: Western Blotting, Immunohistochemistry (Paraffin), ChIP Magnetic
- 5605: Western Blotting, Immunofluorescence
- 7074: Western Blotting
- 7076: Western Blotting
- 11988: Western Blotting, Immunoprecipitation (Agarose), Immunohistochemistry (Paraffin)
- 12166: Western Blotting, Immunoprecipitation (Magnetic), ChIP Magnetic, Chromatin IP-seq
Product Description
Specificity / Sensitivity
Notch1 (D1E11) XP® Rabbit mAb detects intracellular epitopes between 2400 and 2500 amino acids of human Notch1. It recognizes both the full-length (~300 kDa) and the NTM region (~120 kDa). The antibody cannot detect the extracellular (ligand-binding) domain of Notch1 following cleavage at the S2 site by ADAM-type metalloproteases. Cleaved Notch1 (V1744) (D3B8) Rabbit mAb detects endogenous levels of the Notch1 intracellular domain (NICD) only when released by cleavage between Gly1753 and Val1754 (equivalent to Gly1743/Val1744 of murine notch1). The antibody does not recognize full-length Notch1 or Notch1 cleaved at other positions. The size of the NICD varies among cell lines due to mutations in the Notch1 C-terminus. RBPSUH (D10A4) XP® Rabbit mAb recognizes endogenous levels of total RBPSUH protein. MAML1 (D3K7B) Rabbit mAb recognizes endogenous levels of total MAML1 protein. This antibody does not detect MAML2 or MAML3. c-Myc (D84C12) Rabbit mAb recognizes endogenous levels of total c-Myc protein. p21 Waf1/Cip1 (12D1) Rabbit mAb detects endogenous levels of total p21 protein. The antibody does not cross-react with other CDK inhibitors. HES1 (D6P2U) Rabbit mAb recognizes endogenous levels of total HES1 protein. Cyclin D3 (DCS22) Mouse mAb detects endogenous levels of total cyclin D3 protein. The antibody does not cross-react with cyclin D1 or cyclin D2.
Source / Purification
Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro2438 of human Notch1, the Val1754 cleavage site in human Notch1 (equivalent to Val1744 in mouse Notch1), residues surrounding Gln110 of human RBPSUH protein, residues surrounding Asp269 of human MAML1 protein, amino-terminal residues of c-Myc, residues near the carboxy-terminus of human p21, recombinant protein specific to human HES1 protein, residues 241-260 of recombinant human cyclin D3. Antibodies are purified by protein A and peptide affinity chromatography.
Background
Notch proteins (Notch1-4) are a family of transmembrane receptors that play important roles in development and the determination of cell fate (1). Mature Notch receptors are processed and assembled as heterodimeric proteins, with each dimer comprised of a large extracellular ligand-binding domain, a single-pass transmembrane domain, and a smaller cytoplasmic subunit (Notch intracellular domain, NICD) (2). Binding of Notch receptors to ligands of the Delta-Serrate-Lag2 (DSL) family triggers heterodimer dissociation, exposing the receptors to proteolytic cleavages; these result in release of the NICD, which translocates to the nucleus and activates transcription of downstream target genes (3,4). RBPSUH (Recombining Binding Protein, SUppressor of Hairless), is the DNA-binding component of the transcription complex regulated by canonical Notch signaling. Binding of Notch with RBPSUH activates a transcription activation complex that includes Mastermind-like (MAML) proteins, leading to transcriptional activation of Notch target genes (5-7). The NICD binds and activates c-Myc which functions as a transcriptional regulator with roles in various aspects of cell behavior including proliferation, differentiation and apoptosis (8). The tumor suppressor protein p21 Waf1/Cip1 acts as an inhibitor of cell cycle progression. The NICD-RBPSUH complex binds and activates p21 for transcription (15). HES1 (Hairy and Enhancer of Split 1) is one of seven members of the HES family of basic helix-loop-helix (bHLH) transcription factors that is particularly well known as a repressive mediator of the canonical Notch signaling pathway (10). HES1 plays a key role in mediating Notch-dependent T cell lineage commitment (11), and has been reported to be an essential mediator of Notch-induced T cell acute lymphoblastic leukemia (T-ALL) (11,12). The active complex of cyclin D/CDK4 targets the retinoblastoma protein for phosphorylation, allowing the release of E2F transcription factors that activate G1/S-phase gene expression (13). Transcription of cyclin D is in part regulated by the NICD binding to the promoter region of cyclin D (14).
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- Kobayashi, T. and Kageyama, R. (2010) Genes Cells 15, 689-98.
- Wendorff, A.A. et al. (2010) Immunity 33, 671-84.
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