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| 1mg |
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| 5mg |
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| 10mg |
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| Targets |
Arg‑Arg‑AMC acetate is a specific fluorescent substrate for cathepsin B (CTSB), a lysosomal cysteine protease. Cathepsin B functions as both an endopeptidase and a dipeptidyl peptidase, capable of cleaving dipeptide substrates such as Arg‑Arg‑AMC. The dipeptide sequence is specifically recognized by the active site of cathepsin B. Upon cleavage of the amide bond between the arginine residue and the AMC group, the free AMC fluorophore is released, producing a strong fluorescent signal. The substrate is not cleaved by other cathepsins such as cathepsin L under the same conditions, making it relatively selective for cathepsin B.
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| ln Vitro |
Arg‑Arg‑AMC acetate is a fluorogenic substrate for cathepsin B. In the intact peptide, the fluorescence of the AMC group is quenched. Upon enzymatic cleavage by cathepsin B, the AMC group is released, and its fluorescence increases dramatically. The increase in fluorescence (λex=360-380 nm, λem=440-460 nm) is proportional to cathepsin B activity. The substrate is used to measure cathepsin B activity in biochemical assays, cell lysates, and for screening cathepsin B inhibitors. No specific IC50 values for the substrate itself are reported, as it is a substrate, not an inhibitor.
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| ln Vivo |
No in vivo activity data for the substrate are reported. The compound is an in vitro fluorogenic substrate and is not used as a drug in vivo. It can be used in ex vivo assays to measure cathepsin B activity in tissue homogenates or biological fluids from animal models of cancer, arthritis, and neurodegeneration. The substrate is not administered systemically.
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| Enzyme Assay |
The binding and cleavage of Arg‑Arg‑AMC acetate by cathepsin B are measured by standard in vitro fluorometric protease activity assays. Purified human cathepsin B (0.1-10 nM) is activated in activation buffer (e.g., 50 mM sodium acetate, pH 5.5, 2.5 mM DTT, 2 mM EDTA) at 37degC for 5-15 minutes. The activated enzyme is added to a 96‑well plate containing Arg‑Arg‑AMC acetate (1-500 uM) in assay buffer (e.g., 50 mM sodium acetate, pH 5.5, 2.5 mM DTT). The increase in fluorescence (λex=360-380 nm, λem=440-460 nm) is monitored over time at 37degC using a fluorescence plate reader. The initial velocity is calculated from the linear portion of the fluorescence increase. Kinetic parameters (Km, kcat) are determined by varying the substrate concentration. The pH optimum is typically around pH 5.5-6.0.
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| Cell Assay |
For cellular assays, cells expressing cathepsin B (e.g., cancer cell lines, macrophages) are seeded in 96‑well plates. For measurement of intracellular cathepsin B activity, cells are lysed in a buffer (e.g., 50 mM sodium acetate, pH 5.5, 0.1% Triton X‑100, 2.5 mM DTT) and centrifuged. The supernatant is incubated with Arg‑Arg‑AMC acetate (50-200 uM) in assay buffer for 15-60 minutes at 37degC. Fluorescence is measured as above. For measurement of secreted cathepsin B activity, conditioned medium is collected and incubated with the substrate. For inhibitor studies, cells are pre‑treated with a cathepsin B inhibitor (e.g., CA‑074) before lysis. The substrate can also be used in live‑cell imaging if a cell‑permeable version is used.
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| Animal Protocol |
No animal experiments are performed directly on the substrate. For ex vivo measurement of cathepsin B activity, tissues (e.g., tumor xenografts, arthritic joints, brain) are collected from mice or rats, homogenized in lysis buffer, and centrifuged. The clarified supernatant is incubated with Arg‑Arg‑AMC acetate (50-200 uM) in cathepsin B assay buffer (pH 5.5, 2.5 mM DTT) at 37degC for 15-60 minutes. Fluorescence is measured (λex=360-380 nm, λem=440-460 nm). Cathepsin B activity is expressed as relative fluorescence units (RFU)/min/mg protein. This assay is used to assess the efficacy of cathepsin B inhibitors in vivo and to study cathepsin B expression in disease models.
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| ADME/Pharmacokinetics |
Arg‑Arg‑AMC acetate (C22H33N9O4·xC2H4O2, MW: free base approx. 487.55, acetate salt approx. 547.6, purity ≥69% peptide content) is a solid powder. For storage, the powder should be kept at -80degC for up to 2 years or at -20degC for up to 1 year, sealed and protected from light. For in vitro use, stock solutions in water (≥100 mg/mL) can be prepared and stored at -80degC for up to 6 months or at -20degC for 1 month. The acetate salt form improves aqueous solubility. No PK data are applicable.
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| Toxicity/Toxicokinetics |
No specific toxicity data for the substrate are reported. As a research‑grade fluorogenic substrate, it is not intended for human or veterinary use. Standard laboratory safety precautions for handling chemicals should be followed. The AMC fluorophore has low toxicity at the concentrations used in assays (micromolar range).
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| References | |
| Additional Infomation |
Arg‑Arg‑AMC acetate is a fluorogenic substrate for cathepsin B, a lysosomal cysteine protease involved in protein degradation, antigen processing, and tumor cell invasion. Cathepsin B is overexpressed in many human cancers, including glioblastoma, breast, prostate, lung, and colorectal cancers, and its activity correlates with poor prognosis. The Arg‑Arg‑AMC substrate is one of the most commonly used substrates for measuring cathepsin B activity in biochemical and cell‑based assays. It is used for high‑throughput screening of cathepsin B inhibitors as potential anticancer and anti‑arthritis drugs. The compound is for research use only and has no clinical applications.
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| Molecular Formula |
C22H33N9O4.XC2H4O2
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| Molecular Weight |
487.56 (free base)
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| Appearance |
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, 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.) |
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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