| Size | Price | |
|---|---|---|
| 1mg | ||
| Other Sizes |
| Targets |
PEGs
ATP‑PEG8‑Biotin targets the ATP‑binding site of ATP‑dependent enzymes, including protein kinases, ATP synthases, helicases, chaperones and other ATPases. The ATP moiety binds to the active site of the enzyme in a manner similar to native ATP, while the biotin tag allows the enzyme to be captured on streptavidin‑coated surfaces (e.g., plates, beads, sensors). This enables pull‑down assays, kinase activity assays and screening of ATP‑competitive inhibitors. |
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| ln Vitro |
In cell‑free biochemical assays, ATP‑PEG8‑Biotin serves as a substrate for various ATP‑dependent enzymes. For example, protein kinases can transfer the gamma‑phosphate of ATP‑PEG8‑Biotin to peptide or protein substrates, allowing the phosphorylated product to be captured on streptavidin‑coated plates and detected with anti‑phospho‑specific antibodies. The PEG8 spacer reduces steric interference, allowing efficient enzymatic activity (typically 50‑100% of native ATP depending on the enzyme). Detailed kinetic parameters (Km, kcat) vary by enzyme and should be determined experimentally.
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| ln Vivo |
ATP‑PEG8‑Biotin is not used in vivo as a therapeutic agent. It is exclusively a research tool for in vitro biochemical assays. However, ex vivo applications include the use of ATP‑PEG8‑Biotin in cell lysates to pull down ATP‑binding proteins (e.g., kinases, heat‑shock proteins) for proteomic analysis or to measure kinase activity in complex biological samples following immunoprecipitation.
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| Enzyme Assay |
A standard in vitro kinase activity assay using ATP‑PEG8‑Biotin: A 96‑well streptavidin‑coated plate is prepared. In separate tubes, prepare kinase reaction mix containing: 1) recombinant kinase or immunoprecipitated kinase (0.1‑10 ng), 2) ATP‑PEG8‑Biotin (1‑100 uM), 3) peptide or protein substrate (1‑100 uM), 4) kinase buffer (e.g., 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.01% Triton X‑100). Initiate reaction by adding ATP‑PEG8‑Biotin (or pre‑mix all components). Incubate at 30degC for 30‑60 min. Stop reaction with EDTA (10 mM). Transfer the reaction mixture to the streptavidin plate and incubate for 30‑60 min at 25degC to capture biotinylated substrate (if the substrate is not directly biotinylated, the phosphorylated product will be captured via the ATP‑PEG8‑Biotin moiety). Wash plate 3 times with PBS‑T. Add anti‑phospho‑specific detection antibody (e.g., anti‑phospho‑serine/threonine/tyrosine) followed by HRP‑conjugated secondary antibody. Add TMB substrate and measure absorbance at 450 nm. Kinase activity is proportional to the signal.
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| Cell Assay |
A general cell lysate ATP‑binding protein pull‑down assay: Cells (1×10⁷) are lysed in non‑denaturing lysis buffer (e.g., 20 mM Tris‑HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X‑100, protease and phosphatase inhibitors). Lysates are cleared by centrifugation. ATP‑PEG8‑Biotin is added to the lysate at a final concentration of 10‑50 uM and incubated for 1‑2 h at 4degC with gentle rotation. Streptavidin‑agarose or streptavidin‑magnetic beads are added and incubated for an additional 1‑2 h. Beads are washed 3‑5 times with lysis buffer to remove non‑specific binders. Bound proteins are eluted by boiling in SDS‑PAGE sample buffer or by competition with excess free biotin (10 mM) or ATP (10 mM). Eluted proteins are resolved by SDS‑PAGE and identified by western blot (using antibodies against specific ATP‑binding proteins of interest) or by mass spectrometry for unbiased proteomic analysis.
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| ADME/Pharmacokinetics |
ATP‑PEG8‑Biotin is not administered to animals for pharmacokinetic studies. As a biotinylated ATP analogue, it would be rapidly metabolised by ectonucleotidases and other phosphatases in vivo, and the intact compound is unlikely to reach target tissues after systemic administration. Therefore, PK parameters are not typically determined for this reagent. The compound is stable in aqueous buffers at pH 7.0‑8.0 for several hours but should be used fresh or stored as a stock solution at -20degC or -80degC. ATP‑PEG8‑Biotin is a research‑grade reagent and is not intended for human or therapeutic use.
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| Toxicity/Toxicokinetics |
The compound is non‑toxic at the concentrations used in biochemical assays (1‑100 uM). Standard laboratory safety practices (gloves, lab coat) should be used when handling the lyophilised powder or stock solutions. No special toxicity studies have been performed as this is a reagent, not a drug candidate.
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| References | |
| Additional Infomation |
ATP‑PEG8‑Biotin is a versatile tool for studying ATP‑dependent enzymes. The PEG8 spacer provides a 30‑40 Angstrom flexible linker that separates the ATP head group from the biotin tag, minimising steric interference with enzyme active sites. This allows the conjugate to be used as a substrate for many different classes of enzymes, including protein kinases, lipid kinases, ATPases and synthetases. The compound is stable when stored as a lyophilised powder at -20degC protected from moisture. For research use only; not for diagnostic or therapeutic applications. ATP‑PEG8‑Biotin is commonly used in high‑throughput screening (HTS) campaigns to identify ATP‑competitive inhibitors of kinases and other ATP‑binding enzymes.
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| Molecular Formula |
C36H63N8O22P3S
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|---|---|
| Molecular Weight |
1084.91
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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) |
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 | 0.9217 mL | 4.6087 mL | 9.2174 mL | |
| 5 mM | 0.1843 mL | 0.9217 mL | 1.8435 mL | |
| 10 mM | 0.0922 mL | 0.4609 mL | 0.9217 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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