| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
|
||
| 5mg |
|
||
| Other Sizes |
| Targets |
The molecular target of this peptide is cathepsin D (CTSD), a lysosomal aspartic protease that belongs to the pepsin family. Cathepsin D plays a critical role in protein degradation, antigen processing, and apoptosis. It is also implicated in tumor progression and metastasis, as it degrades extracellular matrix components. This FRET peptide is specifically cleaved by cathepsin D between the P1-P1' residues (the phenylalanine-phenylalanine bond in the -Phe-Phe- sequence). When cathepsin D cleaves the peptide, the physical separation of EDANS from DABCYL relieves fluorescence quenching, resulting in a proportional increase in fluorescence intensity, allowing real-time measurement of enzyme activity. The substrate is highly selective for cathepsin D over other proteases such as cathepsin B, L, or pepsin.
|
|---|---|
| ln Vitro |
In vitro, this FRET peptide is not a drug but a sensitive substrate for cathepsin D activity assays. At concentrations of 1-20 uM, the peptide is efficiently cleaved by recombinant human cathepsin D (0.1-10 nM) in a time- and concentration-dependent manner. The Michaelis-Menten constant (Km) for this substrate with cathepsin D is typically in the low micromolar range (e.g., 1-5 uM). The peptide shows negligible auto-hydrolysis and excellent stability in assay buffers, making it suitable for high-throughput screening (HTS) of cathepsin D inhibitors. The fluorescence signal is stable and linear for up to 60 minutes under optimal conditions. The substrate does not inhibit cathepsin D activity at concentrations used in assays.
|
| ln Vivo |
The FRET peptide substrate is not designed for in vivo use; it is an in vitro reagent for enzyme activity measurements. Elevated cathepsin D activity has been detected in Alzheimer‘s disease brains (senile plaques) and in various cancers (breast, colorectal, gastric). Using this FRET peptide, cathepsin D activity can be quantified in tissue lysates, plasma, or cell culture supernatants. However, the intact peptide is not administered to animals, as the FRET pair would be quenched in vivo. The peptide is used as a diagnostic tool in research settings to correlate enzyme activity with disease progression, not as a therapeutic agent. No in vivo efficacy studies are performed with the peptide itself.
|
| Enzyme Assay |
For in vitro cathepsin D activity assays, the FRET peptide is reconstituted in assay buffer (100 mM sodium acetate, pH 4.0-5.5, containing 0.05% Brij-35 or 0.01% Triton X-100). Optimal pH for cathepsin D is 4.5-5.0. A typical reaction mixture (100 uL total volume) contains 1-10 uM FRET peptide substrate and 0.1-10 nM recombinant human cathepsin D in assay buffer. The reaction is initiated by adding the enzyme, incubated at 37degC for 10-60 min, and fluorescence is measured at λex = 336 nm or 340 nm and λem = 490 nm (for EDANS). The increase in fluorescence over time is recorded in a kinetic mode. The initial velocity (V0) is expressed as relative fluorescence units per minute (RFU/min). For inhibitor screening, the test compound is pre-incubated with the enzyme for 10-30 min before substrate addition. IC₅0 values are calculated from dose-response curves. Pepstatin A (10-100 nM) is used as a positive control.
|
| Cell Assay |
For cellular cathepsin D activity assays, cells (e.g., HeLa, MCF-7 breast cancer cells, or primary neurons) are seeded in 96-well plates (1-2×10⁴ cells/well) and cultured for 24-48 h. Cells are then lysed in lysis buffer (50 mM sodium acetate pH 5.0, 0.1% Triton X-100, 150 mM NaCl) containing protease inhibitors (excluding pepstatin A). Lysates are clarified by centrifugation (10,000 × g, 10 min). Equal amounts of protein (10-50 ug) are added to assay buffer containing 10 uM FRET peptide substrate. The reaction is incubated at 37degC for 30-60 min, and fluorescence is measured. The increase in fluorescence is proportional to cathepsin D activity. For inhibition studies, cells are pre-treated with test compounds (1-100 uM) for 24-48 h before lysis, or inhibitors are added directly to the lysate. Cell viability is assessed by MTT assay in parallel.
|
| Animal Protocol |
Not applicable. The FRET peptide is an in vitro biochemical reagent, not an in vivo therapeutic agent. Animal studies are not performed with this peptide. Instead, the compound is used to measure cathepsin D activity in tissue homogenates harvested from animal models of disease. For example, in a mouse model of Alzheimer's disease (APP/PS1 transgenic mice), brain tissue homogenates (hippocampus, cortex) are prepared in lysis buffer, and cathepsin D activity is quantified using the FRET peptide as described above. Elevated activity is observed in AD mouse brains compared to wild-type controls. This data is used to correlate enzyme activity with amyloid plaque load. For cancer studies, tumor xenografts are harvested, and cathepsin D activity is measured to assess the role of the enzyme in tumor progression. No peptide administration is involved.
|
| ADME/Pharmacokinetics |
Not applicable. The FRET peptide is not a drug and is not administered in vivo. Its stability in biological buffers is high; the peptide should be protected from light to prevent photobleaching of the EDANS fluorophore. For long-term storage, the peptide is stored as a lyophilized powder at -20degC. Stock solutions (1-10 mM) can be prepared in DMSO or 50% acetonitrile/water and stored at -20degC or -80degC. Avoid repeated freeze-thaw cycles. The peptide is soluble in DMSO and water. The purity is typically ≥95% by HPLC. The peptide is light-sensitive; wrap tubes in aluminum foil during storage. No pharmacokinetic data is available as the compound is not intended for systemic delivery.
|
| Toxicity/Toxicokinetics |
For the FRET peptide, GHS hazard statements: H315 (Causes skin irritation), H319 (Causes serious eye irritation), H335 (May cause respiratory irritation). Signal word: Warning. Precautionary statements: P261 (Avoid breathing dust/fume/gas/mist/vapors/spray), P280 (Wear protective gloves/protective clothing/eye protection/face protection), P305+P351+P338 (IF IN EYES: Rinse cautiously with water for several minutes). The peptide is not intended for human consumption. Standard safety precautions for handling peptides apply: use PPE (gloves, lab coat, safety goggles), work in a fume hood, avoid inhalation and skin contact. Storage: powder at -20degC for 3 years; in solvent (DMSO) at -80degC for 6 months, -20degC for 1 month. Protect from light.
|
| Additional Infomation |
Ac-Glu-Asp(EDANS)-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Gly-Lys(DABCYL)-Glu-NH2 acetate (Cathepsin D FRET Substrate; CAS# 400716-78-1) is a research-grade fluorogenic peptide substrate. It is not an FDA-approved drug. It is used for measuring cathepsin D activity in biochemical assays, high-throughput screening (HTS) of cathepsin D inhibitors, and for studying the role of cathepsin D in cancer, neurodegeneration (Alzheimer‘s), and other diseases. For research use only, not for diagnostic or therapeutic applications.
|
| Molecular Formula |
C104H146N24O23S.XC2HF3O2
|
|---|---|
| Molecular Weight |
2132.48 (free base)
|
| Related CAS # |
Ac-Glu-Asp(EDANS)-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Gly-Lys(DABCYL)-Glu-NH2; 400716-78-1
|
| Sequence |
Ac-Glu-{Asp(EDANS)}-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Gly-{Lys(DABCYL)}-Glu-NH2Ac-E-{Asp(EDANS)}-KPILFFRLG-{Lys(DABCYL)}-E-NH2
|
| Appearance |
Solid powder
|
| HS Tariff Code |
2934.99.9001
|
| 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)
|
| 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
|
|---|---|
| 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.) |
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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
