| Size | Price | Stock | Qty |
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| Targets |
PEGs
Biotin alkyne targets the biotin-avidin/streptavidin interaction for affinity purification and detection of biomolecules. The biotin moiety binds to streptavidin with extraordinarily high affinity (Kd ∼10-¹⁵ M), enabling efficient pull-down of alkyne-tagged molecules after click conjugation. The alkyne group targets the CuAAC reaction with azide-containing molecules (including azide-modified proteins, peptides, nucleic acids, and small molecules) to form stable triazole linkages. This orthogonal targeting enables selective labeling of biomolecules in complex mixtures. The compound also targets PROTAC synthesis as a PEG-based linker where biotin serves as a reporter or affinity handle. The biotin-avidin interaction is stable under denaturing conditions (e.g., SDS, urea, guanidine-HCl), making it useful for various downstream applications including Western blotting (HRP-streptavidin), ELISA, pull-down assays, and mass spectrometry sample preparation. The alkyne group is bioorthogonal (does not react with naturally occurring functional groups), ensuring high labeling selectivity. The compound is used to study protein-protein interactions, protein localization, and post-translational modifications. |
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| ln Vitro |
The E3 ubiquitin ligase ligand and the target protein ligand are the two distinct ligands found in PROTAC, which are joined by a linker. Specifically, target proteins are degraded by PROTAC through the intracellular ubiquitin-proteasome system [1].
In vitro, Biotin alkyne enables the detection and enrichment of azide-labeled biomolecules. After incorporation of an azide group into target molecules (via metabolic labeling using azido-sugars for glycoproteins, azido-amino acids for proteins, or chemical modification), the alkyne-biotin is conjugated via CuAAC. Biotinylated molecules can then be detected by streptavidin-HRP in Western blot or ELISA, or purified on streptavidin beads for mass spectrometry analysis. In cell lysates or complex biological mixtures, the compound enables specific labeling and enrichment of azide-tagged species with minimal background (due to the bioorthogonal nature of the alkyne-azide reaction). The labeling efficiency is typically >80-90% under optimized conditions. Biotin alkyne has been used to study glycoproteins, glycosylation pathways, protein synthesis, and RNA modifications. The compound itself has no direct cytotoxicity at concentrations used in click chemistry (10-100 uM). In PROTAC applications, PROTACs incorporating the biotin alkyne as part of the linker can be used to visualize or enrich the PROTAC-target protein complex. |
| ln Vivo |
In vivo applications of Biotin alkyne are limited due to the toxicity of copper, which is required for CuAAC reactions. Copper-free click chemistry variants using strain-promoted azide-alkyne cycloaddition (SPAAC) exist, but the standard biotin alkyne requires copper. Therefore, most in vivo studies using biotin alkyne are conducted ex vivo on tissues or lysates after the animals have been administered azide-labeled metabolic precursors (e.g., azidohomoalanine for nascent protein labeling). For PROTAC applications, PROTACs built using biotin-alkyne linkers (or related biotin-containing linkers) have been used in vivo to study target engagement and degradation. For example, a biotinylated PROTAC can be administered to mice, and target proteins can be pulled down from tumor lysates using streptavidin beads to confirm target degradation and PROTAC-target engagement. However, the biotin alkyne itself is not typically administered alone in animal studies. The compound may be used to label azide-tagged biomolecules in zebrafish embryos or other small organisms, where CuAAC conditions (copper(I) salts, often with a copper chelator like THPTA to reduce toxicity) are tolerated at low concentrations. The biotin-avidin interaction is stable in vivo and can be used for imaging applications if a fluorescent streptavidin probe is injected after labeling, but this is not standard.
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| Enzyme Assay |
A CuAAC reaction protocol for labeling azide-modified proteins with Biotin alkyne: In a total volume of 100 uL, mix azide-containing protein (10-100 ug) in PBS (pH 7.4), Biotin alkyne (100 uM final), CuSO4 (1 mM final), THPTA (copper chelator, 2 mM final), and sodium ascorbate (5 mM final). Incubate at room temperature for 1-2 hours. Quench the reaction by adding EDTA (10 mM final) to chelate copper. Remove excess biotin alkyne by dialysis, spin filtration (10-30 kDa MWCO), or size exclusion chromatography. The biotinylated protein can be detected by Western blot with streptavidin-HRP or purified using streptavidin agarose beads (incubate lysate with beads at 4degC for 2-4 hours, wash with PBS + 0.1% Tween-20, elute with 2 mM biotin in PBS or by boiling in SDS sample buffer). For proteins that are sensitive to copper-induced oxidation, a degassed buffer and an inert atmosphere (N2) may be used, or a copper-free click chemistry reagent (e.g., DBCO-biotin) should be substituted.
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| Cell Assay |
Cellular labeling and detection of nascent proteins using Biotin alkyne: Cells (e.g., HeLa, HEK293) are cultured in methionine-free DMEM with 10% dialyzed FBS and supplemented with the methionine analog azidohomoalanine (AHA, 50-100 uM) for 4-24 hours to incorporate azide groups into newly synthesized proteins (replacing methionine). After labeling, cells are washed and lysed in RIPA buffer. Click chemistry is performed on the lysate: lysate (100-500 ug protein) is incubated with Biotin alkyne (50-100 uM), CuSO4 (1 mM), THPTA (2 mM), and sodium ascorbate (5 mM) for 1-2 hours at room temperature. Biotinylated (azide-labeled) proteins are then captured on streptavidin beads, washed extensively, eluted, and analyzed by SDS-PAGE and silver stain or Western blot. This protocol allows visualization of newly synthesized proteins. For subcellular localization studies, cells can be fixed after AHA labeling (with 4% paraformaldehyde), permeabilized (0.1% Triton X-100), and then subjected to click chemistry with Biotin alkyne (50 uM, same copper conditions) followed by detection with fluorescent streptavidin (e.g., Alexa Fluor 488-streptavidin). The signal reveals sites of protein synthesis. For PROTAC studies, cells are treated with a PROTAC that incorporates a biotin-alkyne or biotin-based linker; after treatment, cells are lysed, and pull-down with streptavidin beads can isolate the PROTAC and its bound target proteins for identification by mass spectrometry.
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| Animal Protocol |
Animal studies using Biotin alkyne are typically limited to ex vivo analysis. In a typical nascent protein labeling experiment, mice are injected intraperitoneally with azidohomoalanine (AHA, 25-50 mg/kg) in PBS. After 4-24 hours, mice are euthanized, and tissues (e.g., liver, kidney, brain, tumor) are harvested. Tissue lysates are prepared and subjected to click chemistry with Biotin alkyne (using the same CuAAC protocol as for cell lysates) to biotinylate AHA-labeled (nascent) proteins. Biotinylated proteins are captured on streptavidin beads, eluted, and analyzed by mass spectrometry to quantify protein synthesis rates. This technique is used to study protein turnover, synthesis rates under different physiological conditions, and the effects of drugs on protein synthesis. For PROTAC studies, a PROTAC with a biotin tag (biotin-PROTAC) can be administered to tumor-bearing mice (e.g., 10-30 mg/kg IP, 2-6 hours). Tumors are harvested, lysed, and incubated with streptavidin beads to pull down the PROTAC and its bound target proteins. The target protein is detected by Western blot to confirm target engagement, and potential PROTAC-bound interaction partners can be identified by mass spectrometry. Biotin alkyne is not typically administered directly to animals; instead, the biotin is incorporated into a larger molecule (biotin-PROTAC or biotin-labeled reagent) before administration. The biotin-avidin interaction is stable in vivo, but the streptavidin probe is usually applied ex vivo on lysates, not injected in vivo. The copper required for CuAAC is not suitable for in vivo reactions in mammals due to toxicity, so in vivo labeling is not performed with Biotin alkyne; instead, animals are dosed with azide-labeled precursors, and tissues are processed for CuAAC ex vivo.
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| ADME/Pharmacokinetics |
Biotin alkyne (MW 281 Da) has a calculated logP of approximately 1.5. The biotin moiety is highly hydrophilic, contributing to aqueous solubility. The alkyne group is small, sterically unhindered, and highly reactive with azides under CuAAC conditions. The compound is stable in PBS at pH 7.4 at 4degC for weeks (protected from light). In vivo, the compound is metabolically stable but is cleared rapidly (t½ ∼15-30 minutes) if administered intravenously due to its low molecular weight and hydrophilicity. The alkyne group is not reactive with endogenous functional groups, making it bioorthogonal. The biotin moiety is bound and retained by endogenous biotin-dependent carboxylases (e.g., acetyl-CoA carboxylase, pyruvate carboxylase) if injected in vivo, but this is not a typical application. In standard usage (ex vivo labeling of lysates), pharmacokinetics are not relevant. The biotin-avidin interaction is stable in the presence of detergents (SDS, NP-40, Triton X-100), high salt, and pH 4-10, but is disrupted by boiling in SDS sample buffer (95degC, 5 minutes) or by treatment with 2 mM biotin. This reversible binding (with very high affinity) is exploited for affinity purification and detection. The compound has excellent stability in storage: store at -20degC, protected from light, under inert atmosphere if possible. The melting point is 169-170degC. The compound is a white to off-white solid powder.
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| Toxicity/Toxicokinetics |
Biotin alkyne has low acute toxicity in standard cell culture and ex vivo applications. The compound is not considered hazardous at typical concentrations (≤100 uM). Copper, which is used in the click reaction, is toxic to cells at concentrations >100 uM, but the CuAAC reaction is performed either on lysates (not live cells) or on fixed cells. For live-cell labeling, copper must be used at lower concentrations (50-100 uM) with chelators (THPTA, BTTAA) and is typically limited to short incubations (10-30 minutes). The biotin alkyne itself has no known mutagenicity or reproductive toxicity. Standard chemical safety practices (gloves, lab coat, safety glasses) should be used when handling the solid powder. Avoid inhalation of dust. Alkyne groups are relatively stable but may be reactive under certain conditions (strong oxidizing agents, strong bases). The compound is for research use only and not for diagnostic or therapeutic use. The biotin moiety is a vitamin and is generally recognized as safe (GRAS) in food and supplements at low concentrations. However, the alkyne-substituted compound is not approved for human consumption. Store at -20degC in a tightly sealed container, protected from light. The compound is stable for at least 2 years under recommended storage conditions. Avoid exposure to moisture, as biotin alkyne may be hygroscopic. If skin or eye contact occurs, rinse thoroughly with water. Use in a well-ventilated area (fume hood) when handling the solid powder.
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| References | |
| Additional Infomation |
Biotin alkyne (CAS: 773888-45-2) is a click-chemistry reagent for biotinylation of azide-tagged biomolecules via copper-catalyzed azide-alkyne cycloaddition (CuAAC). Purity is ≥95% (HPLC). The compound appears as a white to off-white solid powder. It is soluble in DMSO and DMF (typically 10-50 mg/mL). Synonyms include Biotin-Alkyne and 5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]-N-prop-2-yn-1-ylpentanamide. It is used in chemical biology for proteomics (identification of newly synthesized proteins via AHA or azidohomoalanine labeling), cell biology (localization of nascent proteins), and glycobiology (labeling of azido-sugar-labeled glycoproteins). The compound is also used as a PROTAC linker for affinity-based detection of PROTAC-target engagement. The biotin tag enables pull-down of PROTACs and their bound proteins from cell lysates. Biotin alkyne is a valuable tool for studying protein-protein interactions, protein synthesis, degradation, and turnover. It is also used in chemical protein synthesis and for preparing biotinylated probes for various assays (e.g., ELISA, Western blot, immunohistochemistry). It is not an approved drug and is for research use only. The compound is stable at -20degC for long-term storage. The melting point is 169-170degC. The molecular formula is C13H19N3O2S. The molecular weight is 281.37 g/mol.
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| Molecular Formula |
C13H19N3O2S
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|---|---|
| Molecular Weight |
281.37
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| Exact Mass |
281.12
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| CAS # |
773888-45-2
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| PubChem CID |
89434015
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| Appearance |
White to off-white solid powder
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| Density |
1.185±0.06 g/cm3(Predicted)
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| Boiling Point |
614.8±50.0 °C(Predicted)
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| Melting Point |
169-170 °C
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| LogP |
1.51
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
19
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| Complexity |
404
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| Defined Atom Stereocenter Count |
3
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| SMILES |
O=C(NCC#C)CCCC[C@@H]1SC[C@]([C@]1([H])N2)([H])NC2=O
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| InChi Key |
JJXUHRONZVELPY-NHCYSSNCSA-N
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| InChi Code |
InChI=1S/C13H19N3O2S/c1-2-7-14-11(17)6-4-3-5-10-12-9(8-19-10)15-13(18)16-12/h1,9-10,12H,3-8H2,(H,14,17)(H2,15,16,18)/t9-,10-,12-/m0/s1
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| Chemical Name |
5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]-N-prop-2-ynylpentanamide
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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) |
DMSO :~250 mg/mL (~888.51 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (7.39 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 20.8 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: ≥ 2.08 mg/mL (7.39 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 20.8 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: ≥ 2.08 mg/mL (7.39 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 | 3.5540 mL | 17.7702 mL | 35.5404 mL | |
| 5 mM | 0.7108 mL | 3.5540 mL | 7.1081 mL | |
| 10 mM | 0.3554 mL | 1.7770 mL | 3.5540 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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