| Size | Price | Stock | Qty |
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| 1mg |
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| Other Sizes |
| Targets |
GSK-3
Phospho-Glycogen Synthase Peptide-2 is a substrate for glycogen synthase kinase-3 (GSK-3) [28L6-L7][28L10-L11]. GSK-3 is a proline-directed serine-threonine kinase that plays a key role in multiple signaling pathways, including insulin signaling, Wnt signaling, and cell fate determination. The peptide does not target a traditional receptor; rather, it serves as a surrogate substrate for the enzyme. The phosphorylated serine residue in the peptide sequence is a recognition motif for GSK-3, which then phosphorylates a nearby serine or threonine residue in the substrate. This peptide can also be used for the affinity purification of other protein-serine kinases. In vitro, the peptide is a validated substrate for GSK-3, allowing the quantitative measurement of kinase activity in a cell-free system. The peptide is not known to have any intrinsic biological activity in cells unless delivered into the cytoplasm via a transfection reagent or a cell-penetrating peptide; it is used primarily as an in vitro tool in cell lysates. |
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| ln Vitro |
In vitro, Phospho-Glycogen Synthase Peptide-2 acts as a specific substrate for GSK-3. When the peptide is incubated with purified GSK-3 and gamma-32P-ATP, the incorporation of radioactive phosphate into the peptide can be measured, providing a quantitative readout of GSK-3 activity. The peptide is also used in non-radioactive formats, such as ADP-Glo™ assays or ELISA-based assays, to measure GSK-3 activity. The peptide can be used for the affinity purification of protein-serine kinases from cell lysates by immobilizing the peptide on a solid support (e.g., agarose beads) and using it as a bait to pull down kinases that recognize and bind to the phosphorylated substrate sequence.
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| ln Vivo |
Phospho-Glycogen Synthase Peptide-2 is not intended for in vivo studies, as it is an in vitro substrate that does not cross cell membranes efficiently without additional modification. No specific in vivo data are available. It is used exclusively as a biochemical tool in cell-free and cell lysate-based assays to measure GSK-3 activity. For in vivo applications, other methods (e.g., measuring phosphorylation of endogenous GSK-3 substrates by Western blot) are more appropriate.
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| Enzyme Assay |
A typical GSK-3 kinase assay protocol is described. The reaction mixture (25-50 microL) contains: kinase buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 5 mM DTT, 0.1% BSA), 50 microM ATP (with or without gamma-32P-ATP, 0.1-0.5 microCi/microL), and the peptide substrate at a concentration of 100-500 microM. The reaction is initiated by the addition of 0.5-1 microg of recombinant human GSK-3beta (or GSK-3alpha). After incubation at 30degC for 15-30 minutes, the reaction is terminated by spotting an aliquot onto P81 phosphocellulose paper or by adding 0.5% phosphoric acid. The papers are washed 3 times in 0.75% phosphoric acid, rinsed with acetone, and counted in a scintillation counter. For non-radioactive assays, the ADP-Glo™ kinase assay kit (Promega) is used: after the kinase reaction, the remaining ATP is converted to light, and luminescence is measured. The amount of ADP generated is proportional to the kinase activity. For IC50 determination of a GSK-3 inhibitor, the compound is pre-incubated with the enzyme for 10-15 minutes before the addition of the peptide and ATP. The IC50 is calculated by fitting the inhibition curve. The peptide is also used for affinity purification. The peptide is immobilized onto a suitable resin (e.g., via an N-terminal cysteine or biotin-streptavidin). Cell lysates are pre-cleared and then incubated with the immobilized peptide. After washing, bound kinases are eluted and identified by mass spectrometry.
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| Cell Assay |
Cell-based assays are not typically performed with this peptide, as it is not cell-permeable. However, the peptide can be used to measure GSK-3 activity in cell lysates. Cells (e.g., HEK293, HeLa) are lysed in RIPA buffer or a non-denaturing lysis buffer with protease and phosphatase inhibitors. The lysates are cleared by centrifugation. Protein concentration is determined (BCA assay). 10-50 ug of lysate protein is added to a reaction mixture containing the peptide substrate and ATP (as described above for the in vitro assay). The mixture is incubated at 30degC for 30-60 minutes. The incorporation of phosphate into the peptide is detected using a phospho-specific antibody in an ELISA format, or by adding the reaction mixture to a streptavidin-coated plate if a biotinylated version of the peptide is used, followed by detection with a phospho-specific antibody. This allows the measurement of endogenous GSK-3 activity in the lysate. Alternatively, the ADP-Glo™ assay can be used with the lysate. The GSK-3 activity can be normalized to the total protein concentration.
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| Animal Protocol |
No in vivo animal protocols exist for this peptide, as it is not used in animals.
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| ADME/Pharmacokinetics |
Pharmacokinetics are not applicable as the peptide is an in vitro tool and is not administered to animals.
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| Toxicity/Toxicokinetics |
No toxicity data are available for this peptide. As a substrate for kinases, it is not expected to be toxic in vitro at the concentrations used (typically 100-500 uM in the reaction mixture). Standard laboratory safety precautions should be followed.
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| References | |
| Additional Infomation |
Phospho-Glycogen Synthase Peptide-2 (substrate) TFA is a research-grade peptide and is not approved for clinical use. It is a peptide substrate for glycogen synthase kinase-3 (GSK-3) and can be used for affinity purification of protein-serine kinases. The peptide contains a phosphorylated serine residue, making it a substrate for GSK-3. This product is for research use only and not for human therapeutic applications. Store as a lyophilized powder at -20degC in a dry, dark environment.
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| Molecular Formula |
C125H192F3N40O50P
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|---|---|
| Molecular Weight |
3143.07
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| Related CAS # |
Phospho-Glycogen Synthase Peptide-2(substrate);851366-97-7
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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 :~100 mg/mL (~31.82 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 100 mg/mL (31.82 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 | 0.3182 mL | 1.5908 mL | 3.1816 mL | |
| 5 mM | 0.0636 mL | 0.3182 mL | 0.6363 mL | |
| 10 mM | 0.0318 mL | 0.1591 mL | 0.3182 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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