| 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-1
Caspase-1 (Interleukin-1beta converting enzyme, ICE). |
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| ln Vitro |
Rules(This is the suggested procedure. This protocol should be customized to meet your unique requirements, as it just offers guidelines. Assay for Caspase-1 Fluorogenic Activity[1]: 1. Follow your standard protocol for cell culture. 2. After 24 hours, cells that have been transfected transiently with caspase-1 in conjunction with pcDNA3.1, Myc-cIAP2, Myc-cIAP1, or Myc-XIAP are harvested. 3. For cleavage experiments, cells were lysed in CHEGG buffer and then sonicated for 15 seconds at 60% amplitude. 4. 10 mM of the fluorogenic caspase-1 substrate Ac-WEHD-AFC TFA was added to the lysates during incubation. 5. The release of free AFC was measured as arbitrary fluorescence units per minute and tracked continuously for one hour (excitation at 380 nm and emission at 460 nm) at one-minute intervals.
The Ac-WEHD-AFC peptide is specifically recognized and cleaved by active caspase-1. Cleavage occurs at the C-terminal side of the aspartic acid (D) residue, separating the AFC (7-Amino-4-trifluoromethylcoumarin) fluorophore from the peptide. This relieves the quenching effect, resulting in a strong increase in fluorescence, which directly correlates with caspase-1 enzymatic activity. |
| ln Vivo |
Ac-WEHD-AFC itself is not a therapeutic with in vivo activity; it is a substrate. However, it is used in animal model tissue lysates to measure in vivo caspase-1 activity. Increased fluorescence in samples from disease models (e.g., sepsis, autoinflammatory diseases) confirms activation of the inflammasome pathway in that tissue.
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| Enzyme Assay |
In a standard non-cellular assay, purified recombinant human caspase-1 is diluted in assay buffer (e.g., 50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 10 mM DTT). The Ac-WEHD-AFC TFA substrate is added to the reaction. Fluorescence (Ex 400 nm, Em 505 nm) is measured kinetically in a microplate reader at 37degC for 30-60 minutes. The initial linear slope is used to calculate enzyme activity (RFU/min).
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| Cell Assay |
THP-1 human monocytes are seeded in 96-well plates and differentiated into macrophages with PMA. They are then primed with LPS (1 ug/mL, 3h) and treated with ATP (5 mM, 30 min) to activate the NLRP3 inflammasome. Cells are lysed, and the lysate is incubated with Ac-WEHD-AFC TFA. Fluorescence is read to quantify caspase-1 activity.
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| Animal Protocol |
Mice are injected with LPS (e.g., 10 mg/kg, IP) to induce septic shock and caspase-1 activation. After 6 hours, the liver and spleen are harvested. Tissues are homogenized in lysis buffer, and protein concentration is measured. Equal amounts of protein lysate are incubated with Ac-WEHD-AFC TFA, and fluorescence is measured to compare caspase-1 activity between treatment groups.
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| ADME/Pharmacokinetics |
PK properties are not applicable as this is a substrate, not a drug. It is used ex vivo in lysates. The AFC dye is cell-impermeable, but the peptide is designed for use in cell lysates or biochemical assays, not for in vivo administration as a therapeutic.
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| Toxicity/Toxicokinetics |
No toxicity studies are performed for this fluorogenic substrate. Standard laboratory safety precautions apply. The AFC cleavage product is fluorescent and can be measured at low concentrations. The compound itself is not cytotoxic or intended for in vivo use at high concentrations.
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| References | |
| Additional Infomation |
Caspase-1 is a critical mediator of the inflammatory response, responsible for cleaving pro-IL-1beta and pro-IL-18 into their active, secreted forms. This process is a hallmark of inflammasome activation. Ac-WEHD-AFC TFA is a widely used molecular probe for characterizing caspase-1 inhibitors in high-throughput screening (HTS) and for studying the role of pyroptosis and inflammation in various disease models. It is for research use only.
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| Molecular Formula |
C40H38F6N8O13
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|---|---|
| Molecular Weight |
952.77
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| Related CAS # |
Ac-WEHD-AFC;210344-99-3
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| Appearance |
Off-white to light yellow solid powder
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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) |
DMSO :~100 mg/mL (~104.96 mM)
H2O :~50 mg/mL (~52.48 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (2.62 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (2.62 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (2.62 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.0496 mL | 5.2479 mL | 10.4957 mL | |
| 5 mM | 0.2099 mL | 1.0496 mL | 2.0991 mL | |
| 10 mM | 0.1050 mL | 0.5248 mL | 1.0496 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.
Products are for research use only; We do not sell to patients
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