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| Targets |
Protein kinase A (PKA), also known as cAMP-dependent protein kinase. CREBtide is a synthetic substrate for PKA with a Km of 3.9 microM. The peptide is based on the phosphorylation sequence in delta-CREB, which is the natural substrate of PKA. It serves as a specific substrate for PKA and can also be used as a substrate for PKC (protein kinase C). The peptide's sequence includes the canonical PKA phosphorylation motif (Arg-Arg-X-Ser).
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| ln Vitro |
delta-CREB is a splice variant of cAMP response element binding protein (CREB). CREBtide (KRREILSRRPSYR), a synthetic peptide based on the delta-CREB phosphorylation sequence. δ-CREB and CREBtide were explored as substrates for cAMP-dependent protein kinase (cAK). The apparent Km of CREBtide for cAK phosphorylation is 3.9 μM, which is 10 times lower than the synthetic peptide substrate Kemptide (Km=39 μM) most typically employed for cAK assays. The Vmax value of CREBtide is 12.4 mumol/(min.mg) and that of Kemptide is 9.8 mumol/(min.mg). The apparent Km of CREBtide phosphorylated by cGMP-dependent protein kinase (cGK) is 2.9 μM, and the Vmax value is 3.2 mumol/(min.mg). Compared to cAK, cGK phosphorylates delta-CREB and CREBtide significantly more slowly, implying that this peptide retains the high cAK/cGK selectivity displayed by delta-CREB [2].
In vitro, CREBtide is used as a substrate in PKA kinase activity assays. The peptide is phosphorylated by PKA at the serine residue within the recognition sequence. Phosphorylation can be detected using radioactive [gamma-33P]-ATP or [gamma-32P]-ATP in kinase assays, followed by separation of phosphorylated peptide from unincorporated ATP. Alternatively, non-radioactive methods such as fluorescence polarization, AlphaScreen, or ELISA can be used to detect peptide phosphorylation. The peptide's Km of 3.9 microM makes it a suitable substrate for kinetic studies of PKA. |
| ln Vivo |
In vivo studies of CREBtide are not typically performed as the peptide is used as a biochemical tool in in vitro kinase assays rather than as a therapeutic agent. The peptide's primary application is in cell-free and cell-based kinase activity assays to measure PKA activity in cell lysates or purified enzyme preparations. It is not used for in vivo administration in animal models.
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| Enzyme Assay |
In vitro kinase assays using CREBtide measure PKA activity. The assay is performed in kinase buffer containing PKA enzyme (purified or in cell lysates), ATP (with or without radioactive label), and CREBtide substrate at varying concentrations. The reaction is incubated at 30degC for 10-30 minutes. For radioactive assays, the reaction is spotted onto phosphocellulose paper, washed to remove unincorporated ATP, and counted by scintillation. For non-radioactive assays, phosphorylation is detected using phospho-specific antibodies or by mass spectrometry. Km and Vmax values are calculated from Michaelis-Menten kinetics.
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| Cell Assay |
In vitro cell-based assays using CREBtide measure PKA activity in cell lysates. Cells are treated with stimuli that activate or inhibit PKA (e.g., forskolin to activate adenylyl cyclase, or H-89 to inhibit PKA). Cells are lysed in kinase lysis buffer, and cleared lysates are incubated with CREBtide substrate and ATP in kinase buffer. Phosphorylation of CREBtide is detected as described above. The assay can be used to measure changes in PKA activity in response to pharmacological treatments or genetic manipulations. The peptide's sequence includes the PKA phosphorylation motif (Arg-Arg-X-Ser).
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| Animal Protocol |
In vivo animal studies are not performed with CREBtide as it is used as a research tool in biochemical assays. The peptide is not intended for therapeutic use or in vivo administration. Its applications are limited to in vitro kinase activity measurements and related biochemical studies.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of CREBtide are not applicable as the peptide is used as an in vitro research tool rather than as a therapeutic agent. With a molecular weight of 1716.99, the peptide is soluble in aqueous solutions and is typically stored as a lyophilized powder at -20degC. As a synthetic peptide substrate, pharmacokinetic studies are not performed.
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| Toxicity/Toxicokinetics |
Toxicological data for CREBtide are not applicable as the peptide is used as an in vitro research tool and is not administered to animals or humans. Standard safety precautions for handling peptides apply. The compound is intended for research use only and not for human therapeutic use.
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| References |
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| Additional Infomation |
CREBtide is a research-grade synthetic peptide used as a substrate for PKA in kinase activity assays. It is a 13-amino-acid peptide based on the phosphorylation sequence in delta-CREB with the sequence KRREILSRRPSYR. The peptide has a Km of 3.9 microM for PKA. Its molecular formula is C73H129N29O19 with a molecular weight of 1716.99. CREBtide can also serve as a substrate for PKC. It is used in biochemical research to measure PKA activity in cell lysates and purified enzyme preparations. Not approved for clinical use.
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| Molecular Formula |
C73H129N29O19
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|---|---|
| Molecular Weight |
1716.98827338219
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| Exact Mass |
1716.001
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| CAS # |
149155-45-3
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| PubChem CID |
25092394
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| Appearance |
White to off-white solid powder
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| Density |
1.5±0.1 g/cm3
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| Index of Refraction |
1.673
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| LogP |
-6.56
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| Hydrogen Bond Donor Count |
28
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| Hydrogen Bond Acceptor Count |
26
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| Rotatable Bond Count |
59
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| Heavy Atom Count |
121
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| Complexity |
3510
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| Defined Atom Stereocenter Count |
14
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| SMILES |
CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCCN)N
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| InChi Key |
QKXYCULATRFTIQ-KPXMCYQPSA-N
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| InChi Code |
InChI=1S/C73H129N29O19/c1-5-39(4)55(101-60(112)46(25-26-54(106)107)94-58(110)44(16-9-29-87-70(78)79)92-57(109)43(15-8-28-86-69(76)77)91-56(108)42(75)14-6-7-27-74)66(118)98-49(34-38(2)3)61(113)99-51(36-103)63(115)93-45(17-10-30-88-71(80)81)59(111)95-47(18-11-31-89-72(82)83)67(119)102-33-13-20-53(102)65(117)100-52(37-104)64(116)97-50(35-40-21-23-41(105)24-22-40)62(114)96-48(68(120)121)19-12-32-90-73(84)85/h21-24,38-39,42-53,55,103-105H,5-20,25-37,74-75H2,1-4H3,(H,91,108)(H,92,109)(H,93,115)(H,94,110)(H,95,111)(H,96,114)(H,97,116)(H,98,118)(H,99,113)(H,100,117)(H,101,112)(H,106,107)(H,120,121)(H4,76,77,86)(H4,78,79,87)(H4,80,81,88)(H4,82,83,89)(H4,84,85,90)/t39-,42-,43-,44-,45-,46-,47-,48-,49-,50-,51-,52-,53-,55-/m0/s1
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| Chemical Name |
(4S)-5-[[(2S,3S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[(2S)-2-[[(2S)-1-[[(2S)-1-[[(1S)-1-carboxy-4-(diaminomethylideneamino)butyl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]carbamoyl]pyrrolidin-1-yl]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-4-[[(2S)-2-[[(2S)-2-[[(2S)-2,6-diaminohexanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-5-oxopentanoic acid
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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 : ~100 mg/mL (~58.24 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 50 mg/mL (29.12 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
(Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.5824 mL | 2.9121 mL | 5.8241 mL | |
| 5 mM | 0.1165 mL | 0.5824 mL | 1.1648 mL | |
| 10 mM | 0.0582 mL | 0.2912 mL | 0.5824 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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