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| Targets |
Glycogen synthase kinase-3 (GSK-3) alpha and beta isoforms. The GSK3 Substrate peptide is a substrate for both GSK-3alpha and GSK-3beta, which are serine/threonine protein kinases involved in diverse cellular processes including glycogen metabolism, cell cycle regulation, apoptosis, neuronal development, and insulin signaling. GSK-3 phosphorylates its substrates on specific serine/threonine residues, and the peptide substrate contains a consensus sequence recognized by GSK-3. When incubated with GSK-3 in the presence of ATP, the peptide becomes phosphorylated, and the incorporation of phosphate can be quantified as a measure of kinase activity. This peptide is not a drug but a research reagent for kinase activity measurement.
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| ln Vitro |
In vitro, GSK3 Substrate, alpha, beta subunit is used as a standard substrate to measure GSK-3 activity. The peptide is phosphorylated by purified recombinant GSK-3alpha or GSK-3beta in a kinase reaction containing ATP and appropriate buffer conditions. The phosphorylation efficiency is typically dose-dependent with respect to enzyme concentration and time. This peptide can be used to determine the specific activity of GSK-3 preparations, to compare the activity of GSK-3alpha versus GSK-3beta isoforms, or to evaluate the inhibitory potency of GSK-3 inhibitors in enzymatic assays. The peptide's sequence (RAAVPPSPSLSRHSSPHQSEDEEE) contains multiple serine residues that serve as phosphorylation sites for GSK-3. Unlike many protein substrates, this peptide is not used to assess biological functions such as cell proliferation or differentiation, but solely as an enzymatic tool.
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| ln Vivo |
Not applicable (research reagent, not a drug). The GSK3 Substrate, alpha, beta subunit peptide is not administered to animals as a therapeutic agent and is not used in in vivo efficacy studies. It is a biochemical reagent designed exclusively for in vitro enzymatic assays. Therefore, in vivo activity data such as absorption, distribution, efficacy, or toxicity in animal models are not available or relevant for this product. Some researchers may use the peptide as a standard for detecting GSK-3 activity in tissue lysates, but the peptide itself is not injected into animals.
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| Enzyme Assay |
For a standard GSK-3 kinase assay using the peptide substrate, the reaction mixture (30-50 uL) contains recombinant GSK-3alpha or GSK-3beta (0.1-1 ug), GSK3 Substrate peptide (50-200 uM), ATP (50-200 uM), and 5-10 uCi of [gamma-33P]ATP (or [gamma-32P]ATP) in kinase assay buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.1% NP-40). After incubation at 30degC for 30-60 minutes, the reaction is terminated by adding 20 uL of 0.5% phosphoric acid. The mixture is spotted onto P81 phosphocellulose filter papers. Filters are washed 3 times with 0.5% phosphoric acid (5 min each), dried, and the incorporated radioactivity is quantified by liquid scintillation counting. For non-radioactive assays, a homogeneous time-resolved fluorescence (HTRF) or AlphaScreen assay can be used, employing a biotinylated peptide substrate and detection of phosphorylated product with a specific antibody. For inhibitor studies, the compound (e.g., lithium chloride, SB-216763, CHIR99021) is pre-incubated with GSK-3 for 10 minutes before adding ATP and peptide substrate. The percent inhibition is calculated relative to the DMSO control, and IC50 values are determined from dose-response curves using nonlinear regression.
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| Cell Assay |
For cell-based GSK-3 activity assays, cells are lysed in kinase lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM DTT, protease and phosphatase inhibitors). After centrifugation (15,000 × g, 15 min, 4degC), supernatants are collected, and protein concentration is quantified. For immunoprecipitation assays, 200-500 ug of cell lysate is incubated with anti-GSK-3 antibody (1-2 ug) and protein A/G agarose beads overnight at 4degC. Beads are washed twice with lysis buffer and twice with kinase buffer. The immunoprecipitated GSK-3 is then incubated with the peptide substrate (50-100 uM) and ATP (50-200 uM) in kinase buffer (30 uL final volume) for 30 minutes at 30degC. The reaction is terminated and processed as described for the non-cellular assay. Alternatively, cell lysates can be used directly: 10-50 ug of lysate protein is incubated with the peptide substrate and ATP in kinase buffer for 30-60 min at 30degC. Phosphorylation of the peptide is measured using a phospho-specific antibody in an ELISA format or by mass spectrometry. For normalization, the protein concentration is used.
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| Animal Protocol |
Not applicable. The GSK3 Substrate, alpha, beta subunit peptide is a research reagent for in vitro kinase assays and is not administered to animals. Therefore, in vivo animal experiment protocols using this peptide as a therapeutic agent are not available. The peptide may be used ex vivo: after treating animals with a GSK-3 inhibitor or disease model induction, tissues (e.g., brain, liver) are harvested and homogenized, and GSK-3 activity in tissue lysates is measured using this peptide substrate. In such ex vivo studies, a typical procedure involves homogenizing tissue in lysis buffer, measuring protein concentration, and incubating 20-100 ug of lysate with the peptide substrate (100 uM) and ATP (100 uM) in kinase buffer for 30-60 min at 30degC, followed by quantification of phosphorylated peptide by ELISA, mass spectrometry, or radioactivity.
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| ADME/Pharmacokinetics |
Not applicable (research reagent, not a drug). The GSK3 Substrate, alpha, beta subunit peptide is a biochemical tool for measuring GSK-3 activity in enzymatic assays and is not intended for in vivo administration. Therefore, pharmacokinetic properties such as absorption, distribution, metabolism, excretion (ADME), half-life, bioavailability, or clearance are not relevant or available for this product. The peptide is used only in test tubes or cell lysates, not in living organisms. When used in cell lysates or purified enzyme systems, the peptide is stable under assay conditions and is consumed (phosphorylated) by GSK-3, but this represents enzymatic turnover, not pharmacokinetics. For researchers interested in GSK-3 inhibitor pharmacokinetics, separate studies using drug candidates are required.
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| Toxicity/Toxicokinetics |
Not applicable. The GSK3 Substrate, alpha, beta subunit peptide is a research reagent for in vitro assays and is not administered to animals or humans. As such, toxicity data (e.g., acute toxicity, genotoxicity, organ toxicity, carcinogenicity) are not available and are not relevant for this product. The peptide is composed of naturally occurring amino acids and does not contain toxic chemical groups. When handled according to standard laboratory safety practices, the peptide poses no significant health risk. However, as with all laboratory chemicals, it should be handled with appropriate personal protective equipment (gloves, lab coat, eye protection). The trifluoroacetate (TFA) or acetate counterion present in the peptide preparation is at low levels and is generally considered non-toxic.
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| References | |
| Additional Infomation |
GSK-3 (glycogen synthase kinase-3) is a ubiquitously expressed serine/threonine kinase with two isoforms, alpha and beta, that play critical roles in multiple signaling pathways including Wnt, insulin, PI3K/AKT, and Hedgehog. GSK-3 is a key regulator of glycogen metabolism (phosphorylating and inactivating glycogen synthase), as well as cell cycle progression, apoptosis, neuronal function, and stem cell maintenance. Dysregulation of GSK-3 is implicated in type 2 diabetes, Alzheimer's disease, bipolar disorder, and cancer. The GSK3 Substrate, alpha, beta subunit peptide is a synthetic peptide derived from the phosphorylation site of the glycogen synthase protein or other GSK-3 substrates, containing the consensus motif S/TXXXS/T (where serine/threonine residues are phosphorylated). The peptide sequence is RAAVPPSPSLSRHSSPHQSEDEEE, and it serves as a standardized tool for measuring GSK-3 activity in kinase assays. It is not a drug and is for research use only. The peptide is typically supplied as a lyophilized powder and should be reconstituted in water or buffer before use. This product is not approved for clinical or therapeutic applications.
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| Molecular Formula |
C108H169N35O42
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| Molecular Weight |
2629.90
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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 (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) |
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 | 0.3802 mL | 1.9012 mL | 3.8024 mL | |
| 5 mM | 0.0760 mL | 0.3802 mL | 0.7605 mL | |
| 10 mM | 0.0380 mL | 0.1901 mL | 0.3802 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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