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| Targets |
DAPK (death-associated protein kinase) is a calcium/calmodulin (CaM)-dependent serine/threonine protein kinase that plays a key role in apoptosis, autophagy, and tumor suppression. DAPK Substrate Peptide TFA is not a drug and does not bind to a receptor; rather, it is a peptide sequence recognized and phosphorylated by DAPK. The peptide substrate contains specific amino acid residues (usually an arginine in the -3 position and a hydrophobic residue at the phosphorylation site) that fit into the catalytic pocket of DAPK. DAPK phosphorylates the substrate peptide at a serine or threonine residue, and the reaction can be quantified in vitro.
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| ln Vitro |
The substrate peptide itself has no inherent biological activity; its utility is as a tool to measure DAPK enzymatic activity in vitro. It is used to determine the kinetic parameters (Km, Vmax) of DAPK, to screen for DAPK inhibitors or activators, and to measure DAPK activity in cell lysates or immunoprecipitates. The peptide is recognized by DAPK with a Km of 9 microM, indicating a moderately high affinity for the active site. The TFA salt form is used as the standard substrate in kinase activity assays. No other in vitro “activity” is relevant for a substrate peptide.
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| ln Vivo |
Because DAPK Substrate Peptide TFA is a research tool, it is not administered in vivo. It is used exclusively for in vitro kinase assays and does not have in vivo activity as a therapeutic agent. The substrate peptide itself, if injected, would be rapidly degraded by proteases and would not produce a meaningful biological effect. Its role is confined to biochemical and cell-free experiments to study DAPK function. No in vivo animal studies have been performed with this peptide alone; it is only used as a reagent.
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| Enzyme Assay |
DAPK Substrate Peptide TFA is used in in vitro kinase assays with purified recombinant DAPK (either full-length or catalytic domain). The standard protocol for DAPK activity measurement is as follows: Prepare a kinase reaction buffer containing 50 mM HEPES-NaOH (pH 7.5), 10 mM MgCl2, 2 mM MnCl2, 1 mM DTT, 0.1 mM CaCl2, 0.5 microM calmodulin (CaM), 50 microM ATP (including 1-10 microCi of gamma-32P-ATP or 33P-ATP). DAPK substrate peptide (derived from the sequence, e.g., KKALRRQE FVA S, sequence varies by vendor) is added to the reaction mixture at concentrations ranging from 0 to 200 microM (typically 0, 10, 25, 50, 100, 200 microM). The reaction is initiated by adding DAPK (10-50 ng) to a final volume of 25-50 microL. After incubation at 30degC for 15-30 min, the reaction is stopped by adding 10% trichloroacetic acid (TCA) or by spotting onto P81 phosphocellulose paper. For the P81 paper method, 20 microL of the reaction mixture is spotted onto a 1 cm2 P81 square. The squares are washed 3 times with 75 mM phosphoric acid (10 min each), once with acetone, and dried. Radioactivity (incorporated 32P) is measured by liquid scintillation counting. The amount of incorporated phosphate (pmol) is calculated based on the specific activity of ATP. Kinetic parameters (Km, Vmax) are derived by fitting the Michaelis-Menten equation (velocity vs. substrate concentration) using non-linear regression (e.g., GraphPad Prism). For the substrate peptide, the Km is determined to be 9 uM. The turnover number (kcat) can also be calculated. To measure DAPK activity in complex biological samples (e.g., cell lysates), the substrate peptide can be used after immunoprecipitation of DAPK. For inhibitor screening, test compounds are pre-incubated with DAPK for 10 min before adding the substrate peptide. The IC50 of DAPK inhibitors is then calculated.
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| Cell Assay |
Since the DAPK substrate peptide is not a drug, it is not used in standard cell viability or proliferation assays. However, it can be used in cell lysate-based kinase activity assays. In a typical protocol, cells (e.g., HeLa, HEK-293, or cancer cells) are cultured in 10 cm dishes (80-90% confluent). Cells are washed with cold PBS and lysed in RIPA buffer or a non-denaturing lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM DTT, 1 mM PMSF, and protease inhibitors). Lysates are cleared by centrifugation at 13,000 rpm for 10 min at 4degC. Protein concentration is determined by BCA assay. For each reaction, 50-200 microg of lysate is used. Alternatively, DAPK is immunoprecipitated from cell lysates using a specific anti-DAPK antibody and protein A/G agarose beads. The immunoprecipitated beads are washed 2-3 times with kinase buffer. The DAPK activity assay is then performed as described in the “Enzyme/Receptor Binding” section, using the DAPK Substrate Peptide TFA (50-200 uM) and gamma-32P-ATP (50 uM, 0.5-1 uCi/reaction). After stopping the reaction, the mixture is spotted onto P81 paper, washed, and counted. The activity is expressed as pmol phosphate incorporated per mg lysate per minute (or per immunoprecipitate). To control for the amount of DAPK present, a parallel sample is run with the same lysate for Western blot detection of DAPK protein levels. The DAPK substrate peptide is also used in FRET-based or luminescence-based (ADP-Glo) kinase assays that do not require radioactivity. For ADP-Glo, 10 uL of kinase reaction (40 mM Tris pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA, 50 uM ATP, 200 uM substrate peptide, and 50 ng DAPK) is incubated for 60 min. Then 10 uL of ADP-Glo reagent is added, incubated for 40 min, followed by 20 uL of Kinase Detection Reagent, incubated for 30 min, and luminescence is read. The luminescence signal is proportional to ADP (kinase activity). The DAPK Substrate Peptide TFA is used as the standard substrate in this format.
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| Animal Protocol |
The peptide is a laboratory reagent and is not administered to animals for efficacy studies. However, it can be used to measure ex vivo DAPK activity in tissues harvested from animal models of disease (e.g., tumor xenografts, ischemia-reperfusion injury, models of neurodegeneration). In a typical ex vivo protocol, a mouse or rat is euthanized, and the tissue of interest (e.g., brain, liver, tumor) is rapidly excised, snap-frozen in liquid nitrogen, and stored at -80degC. The frozen tissue is pulverized and homogenized in lysis buffer using a tissue grinder or by sonication. The lysate is cleared by centrifugation. DAPK is immunoprecipitated from the lysate (500-1000 microg total protein) using an anti-DAPK antibody. The immunoprecipitates are washed, and the DAPK activity is measured using the DAPK Substrate Peptide TFA and 32P-ATP as described. Results are compared between different treatment groups (e.g., drug-treated vs. vehicle) to determine the effect of the treatment on DAPK activity in vivo. For pharmacological studies, a test compound (a putative DAPK inhibitor or activator) may be administered to animals (e.g., by oral gavage or i.p. injection) before tissue collection, and the DAPK activity is then measured ex vivo using the substrate peptide. This approach links the molecular effect (change in DAPK activity) to the in vivo treatment. The peptide itself is not used in the live animal.
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| ADME/Pharmacokinetics |
The pharmacokinetics of the peptide are irrelevant because it is a research substrate used only in vitro. It does not reach systemic circulation and has no therapeutic application. The TFA counterion improves the solubility and solid-state stability of the peptide for use as a laboratory reagent. The molecular weight is approximately 1692.84 Da. The peptide is soluble in water or PBS at concentrations up to 10 mg/mL. For stock solutions, the peptide is typically dissolved in water or 50 mM Tris-HCl (pH 7.5) and stored in aliquots at -20degC or -80degC to avoid repeated freeze-thaw cycles. The TFA salt form is stable for 2 years when stored as a dry powder at -20degC, protected from light and moisture. The peptide does not require special handling beyond standard laboratory safety precautions.
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| Toxicity/Toxicokinetics |
The DAPK Substrate Peptide TFA is a synthetic peptide (TFA salt) used solely for research purposes. It is not intended for human or veterinary use. The peptide does not have toxicological properties beyond those of any other peptide-based laboratory reagent. The TFA counterion is the trifluoroacetate anion, which is considered safe at the residual amounts present in the final product (typically <5%). The acute toxicity is negligible; the LD50 is not applicable because the compound is not meant for in vivo administration. When handling, standard precautions should be taken: wear gloves, a lab coat, and safety glasses. Avoid inhalation of the powder and avoid skin contact (though the risk of irritation is low). The compound is not known to be mutagenic, carcinogenic, or developmentally toxic. If accidentally ingested or injected in large quantities (unlikely), medical attention should be sought. For disposal, follow institutional guidelines for non-hazardous chemical waste. The product should be stored in a sealed, dry container at -20degC, away from light and moisture, to maintain stability. DAPK Substrate Peptide TFA is strictly for research use only.
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| References | |
| Additional Infomation |
Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-regulated Ser/Thr protein kinase that is a positive mediator of apoptosis (programmed cell death). DAPK is involved in various cellular processes including apoptosis, autophagy, cytoskeleton reorganization, and tumor suppression. Reduced DAPK expression or activity is observed in many human cancers (e.g., non-small cell lung cancer, breast cancer, bladder cancer, colorectal cancer) due to promoter hypermethylation, and it is associated with poor prognosis and metastatic potential. DAPK is thus considered a tumor suppressor. The DAPK Substrate Peptide is derived from a known DAPK phosphorylation site, typically myosin light chain (MLC) or a synthetic peptide optimized for high affinity. The substrate sequence is often RRFS*V (where S* is the phosphorylatable serine) or similar. For the research-grade peptide, the precise sequence can vary by manufacturer, but the common motif is based on the MLC phosphorylation site. The peptide has a Km of 9 uM, which is a typical value for peptide substrates of Ser/Thr kinases. The TFA (trifluoroacetic acid) salt form is used to convert the peptide from the solid-phase synthesis resin into a stable, soluble, and crystallizable powder. DAPK Substrate Peptide is used for DAPK activity assays, for screening of DAPK inhibitors (e.g., for cancer therapy, as DAPK reactivation might suppress tumor growth) or activators (for neuroprotection). It is also used in drug discovery programs aiming to modulate DAPK activity. The compound is for research use only.
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| Molecular Formula |
C72H116F3N25O19
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| Molecular Weight |
1692.84
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| Related CAS # |
DAPK Substrate Peptide;386769-53-5
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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) |
H2O :~50 mg/mL (~29.54 mM)
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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 | 0.5907 mL | 2.9536 mL | 5.9072 mL | |
| 5 mM | 0.1181 mL | 0.5907 mL | 1.1814 mL | |
| 10 mM | 0.0591 mL | 0.2954 mL | 0.5907 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.
Products are for research use only; We do not sell to patients
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