| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 50mg |
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| 100mg |
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| Other Sizes |
| Targets |
(Rac)-Ibrutinib alkyne targets Bruton‘s tyrosine kinase (BTK). Ibrutinib (and its alkyne analog) bind covalently to cysteine residue 481 (Cys-481) in the active site of BTK. This irreversible binding blocks the kinase activity, preventing B-cell receptor signaling. The alkyne group does not interfere with binding but acts as a tag. This probe is used to pull down BTK (using alkyne-biotin) or to visualize BTK in cells (using alkyne-fluorophore). It also interacts with other kinases (e.g., ITK, TEC, EGFR) due to the presence of a conserved cysteine, making it a broad-spectrum probe for kinases with active-site cysteines.
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| ln Vitro |
In vitro, (Rac)-Ibrutinib alkyne retains the ability to bind and inhibit BTK. In an enzymatic activity assay using purified BTK, the alkyne probe inhibits kinase activity with an IC50 in the single-digit nanomolar range (similar to Ibrutinib). Its primary application, however, is as a reporter. When incubated with cell lysates (10 nM-1 uM), it covalently binds to BTK. The lysates can then be subjected to "click chemistry" with biotin-azide. The biotinylated protein can be captured on streptavidin beads and detected by Western blot using an anti-BTK antibody. This validates target engagement. It is also used to label live cells (1 uM, 1 h), followed by fixation and click reaction with a fluorescent dye for imaging.
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| ln Vivo |
In vivo, (Rac)-Ibrutinib alkyne is not used as a drug but as a probe to study drug distribution. It can be injected into tumor-bearing mice (e.g., 10 mg/kg, IP) to evaluate the biodistribution of BTK occupancy. After a defined period (1-24 h), tissues are harvested, homogenized, and the probe is "clicked" with a fluorophore. Fluorescence imaging or flow cytometry can then be used to identify which cells or tissues the drug has penetrated. Alternatively, it is used to trace protein targets in animal tissues. It is a versatile tool for chemoproteomics.
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| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): For a kinobeads or pull-down assay, incubate 100 uL of cell lysate (1 mg/mL protein) with 1 uM of (Rac)-Ibrutinib alkyne for 1 h at 4degC. Add 10 uM biotin-azide, 50 uM THPTA (ligand), 1 mM CuSO4, and 2 mM sodium ascorbate for 30 min at room temperature (click reaction). Add 30 uL of streptavidin-agarose beads and incubate for 1 h. Wash beads, elute with SDS buffer, and run on a Western blot. Probe with anti-BTK antibody to confirm pulldown. For competition, pre-incubate with unlabeled Ibrutinib (10 uM) to block binding.
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| Cell Assay |
General protocol for in vitro cell-based experiments: Culture Ramos (B-cell lymphoma) cells in RPMI + 10% FBS. Treat live cells with (Rac)-Ibrutinib alkyne (0.1-10 uM) for 1-2 h. Harvest cells, wash to remove unbound probe. Fix cells with 4% PFA for 15 min. Permeabilize with 0.1% Triton X-100. Perform the CuAAC (click) reaction using 5 uM TAMRA-azide (fluorophore) for 30 min. Wash cells and analyze by flow cytometry (PE channel) or image by confocal microscopy (Ex/Em 550/570 nm). Fluorescence intensity indicates the amount of the probe bound to intracellular targets (BTK). For a competition assay, pre-treat cells with 10 uM unlabeled Ibrutinib to block the signal.
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| Animal Protocol |
General protocol for in vivo animal experiments: Use 6-8 week old female BALB/c nude mice bearing xenografts (Ramos cells). Administer (Rac)-Ibrutinib alkyne (10 mg/kg) via intraperitoneal injection. After 2 hours, sacrifice the mice, harvest the tumor tissue, and homogenize in PBS. Centrifuge to pellet debris. Incubate the supernatant with biotin-azide and click chemistry reagents as described. Capture biotinylated proteins on streptavidin beads, and elute for Western blotting. This measures target occupancy in the tumor. Alternatively, treat mice with the probe, then perform ex vivo click chemistry on tissue sections for imaging to visualize drug distribution.
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| ADME/Pharmacokinetics |
General pharmacokinetic properties: (Rac)-Ibrutinib alkyne is a small molecule (MW ~440 g/mol). It has a LogP ~3. It is soluble in DMSO. It is metabolized by CYP3A4. In mice, oral bioavailability is moderate (~40%), with a terminal half-life (t1/2) of 2-4 h. It is highly protein bound (>95%). For storage, it is supplied as a powder and should be stored at -20degC. It is stable in DMSO for several months.
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| Toxicity/Toxicokinetics |
General toxicity profile: The probe is chemically similar to Ibrutinib, which is an approved drug. At concentrations used for labeling (1 uM), it is not cytotoxic to cells. In animal studies, single doses up to 50 mg/kg are generally well-tolerated. However, as a covalent inhibitor, caution should be used, and gloves should be worn. It is not a controlled substance.
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| References | |
| Additional Infomation |
The alkyne group is on the piperidine ring of the Ibrutinib molecule. This probe is part of a class of "clickable" probes used for chemical proteomics. It is a powerful tool for studying drug-protein interactions in complex biological systems. For research use only.
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| Molecular Formula |
C25H22N6O2
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|---|---|
| Molecular Weight |
438.48
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| CAS # |
936563-91-6
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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 |
| 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 (~228.06 mM; with sonication)
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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 | 2.2806 mL | 11.4030 mL | 22.8061 mL | |
| 5 mM | 0.4561 mL | 2.2806 mL | 4.5612 mL | |
| 10 mM | 0.2281 mL | 1.1403 mL | 2.2806 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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