| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
PKA 2.8 nM (Ki)
PKA Inhibitor Fragment (6-22) amide specifically targets the catalytic subunit of cAMP-dependent protein kinase A (PKA). The peptide acts as a pseudosubstrate inhibitor: it binds to the substrate-binding site of the PKA catalytic subunit with high affinity, blocking the access of natural protein substrates. The Ki is 2.8 nM, indicating potent inhibition. The sequence is derived from the active site of PKI, a naturally occurring PKA inhibitor. The C-terminal amidation (instead of a free carboxyl group) enhances the peptide's stability against exopeptidase degradation. The compound exhibits high selectivity for PKA and does not significantly inhibit other protein kinases at concentrations used for PKA inhibition. |
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| ln Vitro |
In vitro, this peptide is a potent PKA inhibitor with a Ki of 2.8 nM in enzyme activity assays using purified PKA catalytic subunit and a peptide substrate (e.g., Kemptide). In cell-free systems, it is used to selectively block PKA activity in lysates or to validate PKA-specific signaling. In vivo, the peptide can significantly reverse low-level morphine antinociceptive tolerance in mice, demonstrating its ability to modulate PKA-dependent processes in the central nervous system.
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| ln Vivo |
In vivo, PKA Inhibitor Fragment (6-22) amide TFA has been shown to significantly reverse low-level morphine antinociceptive tolerance in mice. This indicates that the peptide can access its intracellular target within neurons in the central nervous system when administered appropriately. The compound is used in neuropharmacology research to study the role of PKA in opioid tolerance mechanisms. It can be administered via intracerebroventricular (ICV) injection, intrathecal (i.t.) injection, or systemically if a cell-penetrating sequence is added. Further details on other in vivo applications are not available.
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| Enzyme Assay |
A standard in vitro PKA kinase assay is used to confirm inhibition. The reaction mixture (25-50 microL) contains: 40 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.1 mM ATP (including 0.1 microCi/microL gamma-32P-ATP), 50 microM Kemptide substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly), and 0.5-1 ng of purified PKA catalytic subunit. PKA Inhibitor Fragment (6-22) amide TFA is added at varying concentrations (0.1-1000 nM). The reaction is started by the addition of the enzyme, incubated at 30degC for 10-20 min, and terminated by adding 0.5% phosphoric acid. The reaction mixture is spotted onto P81 phosphocellulose paper squares. The papers are washed 3 times in 0.75% phosphoric acid (5 min each), washed once in acetone, air-dried, and placed in scintillation vials. Radioactivity (32P incorporated into the peptide) is counted. The Ki value (2.8 nM) is determined from the dose-response curve using the Cheng-Prusoff equation. For a non-radioactive format, the ADP-Glo™ or a luminescent kinase assay kit can be used, following the manufacturer's protocol.
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| Cell Assay |
A typical protocol for studying the effect of PKA inhibition on morphine tolerance involves in vivo administration, not an in vitro cell-based assay. However, a cell-based assay to assess PKA activity in neurons can be performed: primary cortical or hippocampal neurons are isolated from E16-E18 rat embryos and cultured for 7-14 days. Neurons are treated with PKA Inhibitor Fragment (6-22) amide TFA (1-20 microM) for 1-4 h, with or without prior treatment with morphine (0.1-10 microM for 24-48 h to induce tolerance). After treatment, cells are lysed in RIPA buffer containing a phosphatase inhibitor cocktail. PKA activity in the lysates is measured using a PKA activity assay kit (e.g., a non-radioactive ELISA-based kit) according to the manufacturer's protocol. Alternatively, the phosphorylation level of a known PKA substrate, such as PKA Substrate (RRXS/T) or CREB (pS133), is assessed by Western blot using a phospho-specific antibody. Cell viability is assessed by MTT assay.
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| Animal Protocol |
The standard in vivo protocol involves morphine tolerance in mice. Male ICR or C57BL/6 mice (25-30 g, n=8-12 per group) are used. Morphine tolerance is induced by subcutaneous (s.c.) injection of morphine (10-20 mg/kg) twice daily for 5-7 days. PKA Inhibitor Fragment (6-22) amide TFA is dissolved in sterile saline, 5% DMSO/PBS, or artificial cerebrospinal fluid (aCSF). The peptide is administered via the intracerebroventricular (ICV) route: mice are anesthetized, and a small hole is drilled in the skull (coordinates: 0.5 mm posterior to bregma, 1.0 mm lateral, 2.5 mm deep). A microsyringe is used to inject 3-5 microL of peptide solution (e.g., 1-10 microg/mouse) 15 min before each morphine injection. Alternatively, intrathecal (i.t.) injection at the L5-L6 level (5 microL, 0.5-5 microg/mouse) can be used. The antinociceptive effect is measured by the tail-flick or hot-plate test 30 min after morphine administration on days 1, 3, 5, and 7. The tail-flick latency (time for the mouse to flick its tail away from a radiant heat source) or hot-plate latency (time to lick the hind paw or jump) is recorded. A cutoff time (e.g., 10-15 s) is used to prevent tissue damage. Tolerance is defined as a significant decrease in antinociceptive response over time. The PKA inhibitor group should show a reversal of this tolerance (i.e., the antinociceptive effect is maintained or significantly higher than the morphine-only control group at day 7).
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| ADME/Pharmacokinetics |
No detailed pharmacokinetic data are available for this peptide. As a 17-amino acid peptide (molecular weight: 1982.08 g/mol for the TFA salt), it is expected to have a very short half-life in circulation (<15 minutes) due to rapid degradation by proteases, and its penetration of the blood-brain barrier is minimal. Therefore, for CNS applications, local administration (ICV or i.t.) is preferred to achieve effective concentrations in the brain. The C-terminal amidation may provide some protection against carboxypeptidases, but the peptide remains susceptible to endopeptidases and aminopeptidases. The TFA salt confers water solubility. No information on oral bioavailability is available.
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| Toxicity/Toxicokinetics |
No toxicity data are available for PKA Inhibitor Fragment (6-22) amide TFA. In animal studies (ICV or i.t. administration at the doses used for morphine tolerance reversal), no overt signs of neurotoxicity (e.g., seizures, motor impairment, or mortality) have been reported. The peptide is derived from an endogenous protein and is generally considered to have low inherent toxicity. Standard laboratory safety precautions should be followed.
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| References |
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| Additional Infomation |
PKA Inhibitor Fragment (6-22) amide TFA is a research-grade peptide and is not approved for clinical use. It is a potent inhibitor of cAMP-dependent PKA with a Ki of 2.8 nM. The C-terminal amide modification enhances peptide stability. The compound significantly reverses low-level morphine antinociceptive tolerance in mice. This product is for research use only and not for human therapeutic applications. Store as a lyophilized powder at -20degC, sealed and protected from moisture. For in vivo use, dissolve in sterile saline, aCSF, or a suitable vehicle.
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| Molecular Formula |
C80H130N28O24.XC2HF3O2
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| Related CAS # |
PKA Inhibitor Fragment (6-22) amide;121932-06-7
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| Appearance |
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 (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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
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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.) |
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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