| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
Caspase-4, Caspase-5, and other proteases with a preference for cleaving after aspartic acid residues.
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| ln Vitro |
This substrate shows high selectivity for specific proteases, particularly caspase-4 and caspase-5, which are involved in the non-canonical inflammasome pathway. Upon cleavage by its target protease, the Ac-IEPD-AMC peptide is hydrolyzed at the C-terminal side of the aspartic acid residue, releasing the fluorescent AMC group.
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| ln Vivo |
The in vivo activity of Ac-IEPD-AMC is used as a measure of protease activation. For example, in models of septic shock or inflammatory diseases, increased cleavage of this substrate in tissue lysates indicates elevated activity of the target proteases. It serves as a valuable tool to study the non-canonical inflammasome pathway in vivo.
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| Enzyme Assay |
The primary non-cellular experimental protocol involves incubating the Ac-IEPD-AMC TFA substrate with a purified recombinant protease in assay buffer (e.g., 50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 10 mM DTT). The reaction is initiated by adding the substrate to a final concentration of 10-100 uM. Fluorescence intensity (Ex 355 nm, Em 460 nm) is measured continuously for 30-60 minutes at 37degC. The initial linear portion of the curve is used to calculate enzyme activity.
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| Cell Assay |
For in vitro cell assays, cells (e.g., HEK293T or THP-1 macrophages) are seeded in culture plates and treated with experimental compounds to induce protease activation. After treatment, cells are lysed in a buffer containing 0.1% Triton X-100. The lysate is incubated with the Ac-IEPD-AMC TFA substrate at 37degC for 1-2 hours. The fluorescence of the released AMC is then measured with a microplate reader.
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| Animal Protocol |
In an animal model, tissues of interest (e.g., liver, spleen, or brain) are harvested from treated or control animals. The tissues are homogenized in a lysis buffer. The protein concentration of the lysates is determined using a BCA assay. Equal amounts of protein are then incubated with the Ac-IEPD-AMC TFA substrate in assay buffer for 1-2 hours at 37degC. Fluorescence is measured to determine the proteolytic activity in the lysate.
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| ADME/Pharmacokinetics |
As a substrate, Ac-IEPD-AMC TFA is not intended to have its own pharmacokinetic profile. The goal of its use is to measure the activity of proteases, not to be a systemic drug. In vivo distribution studies could theoretically be performed, but are uncommon. Its primary "PK" property is its stability in biological matrices before being cleaved by its target.
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| Toxicity/Toxicokinetics |
No toxicity studies have been performed for this biochemical reagent. It is not intended for in vivo use at high doses. When handled in research labs, standard safety precautions are sufficient. The AMC group can interact with DNA, but no acute toxicity is reported.
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| References |
[1]. Scott JI, et, al. A Functional Chemiluminescent Probe for in Vivo Imaging of Natural Killer Cell Activity Against Tumours. Angew Chem Int Ed Engl. 2021 Mar 8;60(11):5699-5703.
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| Additional Infomation |
This substrate is used in high-throughput screening (HTS) to identify novel inhibitors of caspase-4 and caspase-5. The released AMC product has a distinct fluorescence that can be continuously monitored. This product is strictly for research use and is not for diagnostic or therapeutic applications in humans.
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| Molecular Formula |
C32H41N5O11.XC2HF3O2
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|---|---|
| Molecular Weight |
671.69 (free acid)
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| Related CAS # |
Ac-IEPD-AMC;216757-33-4
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| Appearance |
Typically exists as solid at room temperature
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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 (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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.) |
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
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