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| Targets |
Biotin-epoxomicin targets the proteasome, a large multi-catalytic protease complex responsible for degrading ubiquitinated proteins. Specifically, it covalently binds to the catalytically active N-terminal threonine residues of the beta subunits, including the constitutive subunits beta1 (caspase-like), beta2 (trypsin-like), and beta5 (chymotrypsin-like), as well as the immunoproteasome subunits LMP7 (beta5i), MECL1 (beta2i), and LMP2 (beta1i). The epoxomicin moiety is an irreversible inhibitor that forms a morpholino adduct with the threonine hydroxyl. The biotin tag allows for affinity capture using streptavidin-conjugated beads, making it possible to pull down and identify active proteasome subunits from cell lysates or purified preparations. This probe operates within the ubiquitin-proteasome pathway (UPP) and is used to study proteasome composition and activity.
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| ln Vitro |
In vitro, Biotin-epoxomicin does not exhibit a traditional pharmacological activity such as an IC50 value for cell killing; instead, its activity is defined by its ability to covalently label and enrich proteasome subunits. At concentrations of 0.1-10 uM, when incubated with cell lysates or purified 20S proteasome complexes at 37degC for 30-60 minutes, it binds to the active subunits. The labeled subunits can then be captured on streptavidin beads, eluted, and identified by LC-MS/MS or Western blot using subunit-specific antibodies. The probe has been used to demonstrate that epoxomicin binds to LMP7, Z, and MECL1 subunits in the immunoproteasome. No cytotoxicity is typically measured for the probe itself, as it is used in pull-down assays. It is not intended for cell viability assays, but if used in cells, it may inhibit proteasome activity at higher concentrations. DMSO is used as solvent (final ≤0.1%).
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| ln Vivo |
No in vivo activity data are available for Biotin-epoxomicin, as it is designed exclusively as an in vitro research probe for biochemical pull-down experiments. It is not intended for administration to live animals for therapeutic or efficacy studies. However, conceptually, one could treat cells in culture with the probe, then lyse the cells and perform the pull-down to study proteasome target engagement in a cellular context. But the compound itself is not used for in vivo animal dosing because the biotin tag may alter pharmacokinetics and the high-affinity binding to streptavidin is not relevant in vivo. For research use only.
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| Enzyme Assay |
For non-cellular (cell-free) proteasome binding assays, purified 20S proteasome (e.g., from rabbit or human cells, 1-5 ug) is diluted in 50 uL of assay buffer (50 mM Tris-HCl pH 7.4, 20 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.01% NP-40). Biotin-epoxomicin is added from a 1 mM DMSO stock to final concentrations of 0.1, 0.5, 1, 5, and 10 uM (DMSO ≤1%). The mixture is incubated at 37degC for 30-60 minutes. Then, 10 uL of streptavidin-agarose beads (pre-washed 3× with assay buffer) are added, and the mixture is rotated at 4degC for 1-2 hours. Beads are collected by centrifugation (500×g, 2 min) and washed 3-5 times with 500 uL of assay buffer containing 0.1% Triton X-100, followed by 2 washes without detergent. Bound proteins are eluted by boiling in 30 uL of 2× SDS-PAGE loading buffer for 5 minutes. Samples are resolved on 12% SDS-PAGE and either stained with Coomassie Brilliant Blue or transferred to PVDF for Western blotting. For Western blot, antibodies against proteasome subunits (e.g., anti-beta5, anti-LMP7, anti-MECL1) are used. DMSO-only control is included. Positive control: free epoxomicin (10 uM) added along with Biotin-epoxomicin to compete for binding, demonstrating specificity.
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| Cell Assay |
For in vitro cell-based pull-down assays, cells (e.g., HeLa, B16 melanoma, or 293T) are cultured in appropriate medium and harvested at 80-90% confluence. Cells are washed with ice-cold PBS and lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1× protease inhibitor cocktail) on ice for 30 minutes. Lysates are clarified by centrifugation (14,000×g, 15 min, 4degC), and protein concentration is determined by BCA assay. For each pull-down, 500-1000 ug of lysate protein in 500 uL of lysis buffer is incubated with Biotin-epoxomicin at 1-10 uM (from DMSO stock) for 1-2 hours at 37degC with gentle shaking. Then, 20 uL of streptavidin-agarose beads (pre-washed) are added, and the mixture is rotated at 4degC overnight or for 2 hours. Beads are washed 5× with 500 uL of lysis buffer, then 2× with PBS. Bound proteins are eluted by boiling in SDS-PAGE buffer as above. Eluates are analyzed by Western blot for proteasome subunits or by LC-MS/MS for proteome-wide identification. Negative control: DMSO only. Competition control: 10-fold excess of free epoxomicin pre-incubated with lysate for 30 min before adding Biotin-epoxomicin. DMSO concentration should be ≤0.1% in the final reaction to avoid non-specific effects.
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| Animal Protocol |
Not applicable. Biotin-epoxomicin is not used for in vivo animal studies. It is an in vitro biochemical probe for pull-down assays from cell lysates or purified proteins. There are no standard protocols for animal dosing, efficacy, or toxicology for this compound. It is not intended for administration to live animals. For research use only.
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| ADME/Pharmacokinetics |
Not applicable. Biotin-epoxomicin is not a drug candidate; its pharmacokinetic parameters (absorption, distribution, metabolism, excretion, half-life, bioavailability) are not relevant and have not been determined. The compound is used exclusively in test-tube assays. For storage, the lyophilized powder should be kept at -20degC for up to 3 years, sealed and desiccated, away from light and moisture. Once dissolved in DMSO (e.g., 1-10 mM stock), it should be stored in aliquots at -80degC for up to 1 year and avoid repeated freeze-thaw cycles. The molecular weight is 724.95 g/mol. Solubility: DMSO (10 mg/mL), poorly soluble in water.
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| Toxicity/Toxicokinetics |
No toxicity data are available for Biotin-epoxomicin as a research probe. At the concentrations used in pull-down assays (1-10 uM), no acute toxic effects are expected in the context of the assay. However, epoxomicin itself is a potent proteasome inhibitor and can be cytotoxic to cells at concentrations above 100 nM. The biotinylated version likely retains proteasome inhibitory activity, so standard caution should be exercised when handling: avoid inhalation, ingestion, and skin/eye contact; use PPE (gloves, lab coat, safety goggles); work in a chemical fume hood. For research use only-not for human diagnostic or therapeutic applications. Dispose of waste according to local hazardous waste regulations.
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| References | |
| Additional Infomation |
CAS: 1334608-28-4. Molecular formula: C35H60N6O8S, molecular weight 724.95. The compound consists of biotin linked to the epoxomicin sequence Ile-Ile-Thr-Leu-((2R)-2-methyloxiranyl) (IIT-[Leu((2R)-2-methyloxiranyl)]). It is also referred to as biotinylated epoxomicin. Purity: typically ≥95% by HPLC. Storage: powder at -20degC, protect from moisture. The probe is used for affinity purification of active proteasome subunits and for studying immunoproteasome biology. It is a controlled substance in some jurisdictions? Not specified. For research use only. Pathway: Ubiquitin-proteasome. Not for human use.
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| Molecular Formula |
C35H60N6O8S
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| Molecular Weight |
724.95
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| CAS # |
1334608-28-4
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| Sequence |
Biotin-IIT-{Leu((2R)-2-methyloxiranyl)}Biotin-Ile-Ile-Thr-{Leu((2R)-2-methyloxiranyl)}
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| Appearance |
White to off-white 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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.3794 mL | 6.8970 mL | 13.7941 mL | |
| 5 mM | 0.2759 mL | 1.3794 mL | 2.7588 mL | |
| 10 mM | 0.1379 mL | 0.6897 mL | 1.3794 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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