Intracellular Staining by Flow Cytometry
|Detection of Notch‑1 in RPMI 8226 Human Cell Line by Flow Cytometry. RPMI 8226 human multiple myeloma cell line was stained with Mouse Anti-Mouse Notch‑1 PerCP‑conjugated Monoclonal Antibody (Catalog # IC5267C, filled histogram) or isotype control antibody (Catalog # IC002C, open histogram). View our protocol for Staining Intracellular Molecules.|
Notch-1 is a 210-240 kDa type I transmembrane glycoprotein that is one of four Notch homologues involved in developmental processes (1-3). Notch signaling is important for maintaining stem cells and inducing differentiation, especially in the nervous system and lymphoid tissues (2-4). Notch can specify binary cell fates. For example, it promotes T cell over B cell development from a common precursor (2). Mouse Notch-1 is synthesized as a 2531 amino acid (aa) precursor that contains an 18 aa signal sequence, a 1707 aa extracellular domain (ECD) with 36 EGF-like repeats and three Lin-12/notch repeats (LNR), a 21 aa transmembrane (TM) segment and a 785 aa cytoplasmic domain that contains six ankyrin repeats, a glutamine-rich domain and a PEST sequence. The 11th and 12th EGF-like repeats, that bind ligands such as Jagged-1 and -2, plus Delta-like-1, -3, and -4 plus DNER in humans, correspond to aa 412-488 in mouse Notch-1 (5, 6). Within the 36 EGF-like repeats, N-linked and multiple O-linked glycans are known to exist. These include O-fucose,O-glucose, O-GlcNAc and O-xylose based glycans. Although the best studied modification involves fucose, the mammalian system is very complex and unlikely fully explains the facilitation of delta-like signaling with an inhibition of Jagged-induced signaling. O-linked glucose appears to be essential for Notch activation, while O-GlcNAc modifications have unclear functions (7). The Notch-1 receptor undergoes post-translational furin-type proteolytic cleavage, generating a heterodimer through the interaction of a hydrophobic area C-terminal to the LNR on the extracellular region with the transmembrane/cytoplasmic portion (8, 9). Upon ligand binding, additional sequential proteolysis by TNF-converting enzyme (ADAM17) 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 (9, 10). Mouse Notch-1 ECD aa 19-526, which includes the first 13 EGF repeats, shows 94%, 91%, 86% and 79% aa sequence identity with corresponding regions of rat, human, canine, and chicken Notch-1, respectively. This region also exhibits 55-58% aa sequence identity with human Notch-2 and Notch-3.
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