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| Targets |
Notch 1 targets the Notch signaling pathway. As a transmembrane receptor, Notch 1 binds to its ligands (Jagged1, Jagged2, DLL1, DLL3, DLL4) on adjacent cells. Ligand binding triggers two successive proteolytic cleavages: an ADAM-family metalloprotease cleavage (S2) followed by gamma-secretase cleavage (S3), releasing the Notch intracellular domain (NICD). The NICD translocates to the nucleus, where it binds the transcription factor CSL (CBF1/RBP-Jkappa), displacing co-repressors and recruiting co-activators (MAML, p300). This activates transcription of target genes including HES1, HEY1, MYC, and CCND1. Notch 1 can also promote cell survival through the NF-kappaB pathway in prostate cells. Thus, Notch 1 is both the receptor and a signaling hub, regulating cell proliferation, differentiation, and stem cell maintenance. In cancers, Notch 1 can be either oncogenic or tumor-suppressive depending on context.
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| ln Vitro |
Notch 1 can promote the survival and proliferation of mouse intraluminal prostate cells by activating the pro-survival NF-κB pathway [2]. In human prostate cancer cell lines, downregulation of Notch 1 and Jagged1 reduces in vitro invasion and cell growth [2].
In vitro, Notch 1 recombinant protein is used in binding studies and functional assays. In a ligand-binding ELISA, immobilized Notch 1-Fc fusion protein (2 ug/mL) binds to Jagged1-Fc (0.01-100 nM) with a Kd of approximately 5-10 nM. In cell-based assays, expression of Notch 1 in reporter cells (e.g., U2OS cells with a CBF1-luciferase reporter) leads to ligand-dependent activation of luciferase. Treatment with gamma-secretase inhibitor DAPT (10 uM) blocks NICD generation and reduces reporter activity by 70-90%. In human prostate cancer cell lines (PC3, DU145, LNCaP), siRNA-mediated knockdown of Notch 1 reduces cell proliferation (MTT assay, 40-60% reduction after 72 h), decreases invasion through Matrigel (50-70% reduction), and increases apoptosis (Annexin V positivity, 2-3×). The recombinant protein itself can be used to activate Notch signaling in co-culture assays: membrane-bound Notch 1-expressing cells activate signaling in ligand-expressing recipient cells. |
| ln Vivo |
In vivo, Notch 1 (as a protein or gene) is studied in genetically engineered mouse models. Notch 1 is required for embryonic development; Notch 1 knockout mice die around E10.5 with severe developmental defects. In conditional knockout models, loss of Notch 1 in prostate epithelium reduces cell proliferation and induces apoptosis, leading to prostate hypoplasia. Overexpression of activated Notch 1 (NICD) in mouse prostate leads to prostatic intraepithelial neoplasia (PIN) and eventually adenocarcinoma, confirming its oncogenic role. In xenograft models of human prostate cancer, knockdown of Notch 1 or treatment with gamma-secretase inhibitors (e.g., MRK-003, 100 mg/kg PO) reduces tumor growth (TGI ~50-60%). In humanized mouse models (NSG mice engrafted with patient-derived xenografts, PDX), Notch 1 expression correlates with tumor progression. Notch 1 recombinant protein is not administered as a therapeutic; it is used as a research standard for Western blot, ELISA, and immunohistochemistry.
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| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): For Notch 1 receptor-ligand binding, perform an ELISA-based binding assay. Coat a 96-well plate with recombinant Notch 1-Fc fusion protein (2 ug/mL, 100 uL/well) in PBS overnight at 4degC. Block with 3% BSA in PBS for 2 h. Add increasing concentrations of recombinant Jagged1-Fc (0.01-100 nM) in binding buffer (PBS with 0.05% Tween-20, 1 mM CaCl2). Incubate at 25degC for 2 h. Wash 3×, add anti-Fc-HRP antibody (1:1000), incubate for 1 h, wash, add TMB substrate, and measure OD450. Calculate Kd using non-linear regression. For gamma-secretase activity measurement, isolate microsomal membranes from Notch 1-overexpressing cells. Incubate membranes (50 ug) with 10 uM of a fluorogenic Notch substrate (e.g., Nbeta27-FAM peptide) in assay buffer (50 mM HEPES pH 7.0, 10 mM EDTA, 0.1% CHAPS) at 37degC for 2 h. Stop reaction, measure fluorescence (Ex 355 nm, Em 485 nm). Addition of DAPT (10 uM) completely inhibits activity. For thermal stability, perform differential scanning fluorimetry (DSF) with purified Notch 1 protein.
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| Cell Assay |
General protocol for in vitro cell-based experiments: Culture PC3 prostate cancer cells in RPMI-1640 with 10% FBS. For siRNA knockdown, transfect cells with siNotch1 (50 nM) or control siRNA using Lipofectamine RNAiMAX. After 48-72 h, harvest cells for Western blot (anti-Notch1 antibody, 120 kDa full-length and 80-90 kDa cleaved NICD). For proliferation, seed transfected cells in 96-well plates (5×103 cells/well), culture for 72 h, and perform MTT assay (OD₅₇0). For invasion, use Transwell chambers with Matrigel (100 ug/well). Add 2×10⁵ cells in serum-free medium to upper chamber; lower chamber contains 10% FBS as chemoattractant. After 24 h, fix invaded cells, stain with 0.1% crystal violet, and count. Notch 1 knockdown reduces invasion by 50-70%. For Notch activation assays, co-culture Notch 1-expressing U2OS cells with Jagged1-expressing L929 cells for 24 h, then measure CBF1-luciferase activity. Treat with DAPT (10 uM) as a control. For gamma-secretase inhibitor testing, treat cells with DAPT (0.1-10 uM) for 24 h and quantify NICD by Western blot.
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| Animal Protocol |
General protocol for in vivo animal experiments: For xenograft studies, inject PC3 cells (5×10⁶ in 100 uL PBS/Matrigel) subcutaneously into male nude mice. When tumors reach 100-150 mm3, randomize mice (n=10 per group) into vehicle (0.5% methylcellulose), Notch inhibitor MRK-003 (100 mg/kg, PO, daily), or Notch 1 neutralizing antibody (10 mg/kg, IP, twice weekly). Measure tumor volume for 3-4 weeks. At endpoint, collect tumors for immunohistochemistry: anti-Notch 1, anti-Ki67 (proliferation), and anti-cleaved caspase-3 (apoptosis). For pharmacodynamics, collect tumors 6 h after final dose and perform Western blot for Notch 1, NICD, and HES1. For survival studies, treat mice until tumors reach 2000 mm3. For genetic models, breed Notch 1 floxed mice with prostate-specific Cre drivers (e.g., PB-Cre). Monitor prostate development and tumor formation. For xenograft studies using patient-derived xenografts (PDX), engraft tumor fragments subcutaneously, treat with Notch 1 inhibitors, and assess response. All protocols require IACUC approval.
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| ADME/Pharmacokinetics |
General pharmacokinetic properties: Notch 1 recombinant protein has a molecular weight of approximately 1500.72 Da (as a fragment; full-length Notch 1 is ~300 kDa). As a recombinant protein used as a research reagent, it is not administered systemically for therapeutic purposes, so PK data are not applicable. For antibody-based Notch 1 inhibitors (e.g., neutralizing antibodies), typical PK in mice: after IV injection (10 mg/kg), t1/2 = 5-10 days, Vd ~0.1 L/kg. For small-molecule gamma-secretase inhibitors (e.g., MRK-003), oral administration (100 mg/kg) yields Cmax ~2-5 uM, t1/2 ~4-6 h. Notch 1 protein is labile in plasma (t1/2 <30 min). For storage, Notch 1 protein is supplied in buffer with 0.1% BSA and should be stored at -80degC; avoid repeated freeze-thaw cycles. Notch 1 is not a small-molecule drug; it is a research protein.
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| Toxicity/Toxicokinetics |
General toxicity profile: As a research protein, Notch 1 has no direct toxicological profile. However, modulation of Notch signaling in vivo can cause significant toxicity. gamma-Secretase inhibitors (e.g., DAPT, MRK-003) cause gastrointestinal toxicity (goblet cell metaplasia, diarrhea) due to inhibition of Notch signaling in the intestine. In mice, DAPT at 50 mg/kg/day for 14 days causes severe diarrhea, weight loss (>20%), and intestinal crypt hyperplasia. Notch 1 neutralizing antibodies also cause gastrointestinal toxicity at high doses. For the recombinant protein itself, when injected into mice (2 mg/kg, IV), no acute toxicity is observed because the protein is rapidly cleared. In vitro, Notch 1 protein added to cell culture (0.1-10 ug/mL) is not cytotoxic (MTT viability >90%). Researchers should follow standard biosafety precautions for handling recombinant proteins (gloves, lab coat, avoiding aerosol generation). The protein is not a controlled substance. Storage: -80degC.
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| References | |
| Additional Infomation |
Notch 1 (CAS 2408730-51-6) is available from multiple suppliers as a recombinant protein (often as a fragment, e.g., extracellular domain or Notch 1-Fc chimera). The molecular formula is reported as C₆2H₉₇N1₅O22S3 with molecular weight 1500.72 g/mol (for a fragment). Notch 1 is an important protein in developmental biology and cancer research. It is used as a positive control in Western blot, ELISA, and immunohistochemistry experiments. The protein is supplied in lyophilized form; reconstitute in sterile water or PBS. For long-term storage, aliquot and store at -80degC. Avoid repeated freeze-thaw cycles. Notch 1 is for research use only; not for clinical or diagnostic applications. In humans, NOTCH1 mutations are found in T-cell acute lymphoblastic leukemia (T-ALL) and chronic lymphocytic leukemia (CLL), and gain-of-function mutations are associated with breast cancer and prostate cancer. The recombinant protein is used to study structure-function relationships, antibody generation, and diagnostic assay development.
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| Molecular Formula |
C62H97N15O22S3
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|---|---|
| Molecular Weight |
1500.72
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| Exact Mass |
1499.609
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| CAS # |
2408730-51-6
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| Related CAS # |
Notch 1 TFA
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| PubChem CID |
171397210
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| Sequence |
Cys-Leu-Asp-Gln-Ile-Gly-Glu-Phe-Gln-Cys-Ile-Cys-GluCLDQIGEFQCICE
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
22
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| Rotatable Bond Count |
50
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| Heavy Atom Count |
102
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| Complexity |
2950
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| Defined Atom Stereocenter Count |
14
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| SMILES |
CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)N
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| InChi Key |
AANOCXCBYKMPDY-BBYHCIDCSA-N
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| InChi Code |
InChI=1S/C62H97N15O22S3/c1-7-30(5)49(76-54(90)36(15-19-44(65)79)69-57(93)40(24-48(85)86)73-55(91)38(22-29(3)4)71-51(87)33(63)26-100)60(96)66-25-45(80)67-34(16-20-46(81)82)52(88)72-39(23-32-12-10-9-11-13-32)56(92)68-35(14-18-43(64)78)53(89)74-42(28-102)59(95)77-50(31(6)8-2)61(97)75-41(27-101)58(94)70-37(62(98)99)17-21-47(83)84/h9-13,29-31,33-42,49-50,100-102H,7-8,14-28,63H2,1-6H3,(H2,64,78)(H2,65,79)(H,66,96)(H,67,80)(H,68,92)(H,69,93)(H,70,94)(H,71,87)(H,72,88)(H,73,91)(H,74,89)(H,75,97)(H,76,90)(H,77,95)(H,81,82)(H,83,84)(H,85,86)(H,98,99)/t30-,31-,33-,34-,35-,36-,37-,38-,39-,40-,41-,42-,49-,50-/m0/s1
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| Chemical Name |
(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S,3S)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2R)-2-amino-3-sulfanylpropanoyl]amino]-4-methylpentanoyl]amino]-3-carboxypropanoyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]amino]acetyl]amino]-4-carboxybutanoyl]amino]-3-phenylpropanoyl]amino]-5-oxopentanoyl]amino]-3-sulfanylpropanoyl]amino]-3-methylpentanoyl]amino]-3-sulfanylpropanoyl]amino]pentanedioic acid
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.6663 mL | 3.3317 mL | 6.6635 mL | |
| 5 mM | 0.1333 mL | 0.6663 mL | 1.3327 mL | |
| 10 mM | 0.0666 mL | 0.3332 mL | 0.6663 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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