FRATtide , CAS No.251087-38-4

CAS: 251087-38-4 Cat. No.: F1448880 Formula: C₁₉₂H₃₂₇N₆₇O₆₀ Molecular Weight: 4534.06

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Overview

FRATtide is a peptide derived from the GSK-3 binding protein that inhibits the phosphorylation of Axin and β-catenin. FRATtide inhibits GSK-3 binding to Axin.

Specifications

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

This product requires cold chain shipping. Ground and other economy services are not available.

Action Type

INHIBITOR

Names and Identifiers

Molecular Weight

4534.06

Application Protocols

No vendor-tested immunoassay protocols (WB, IHC, IF, FC) are applicable to this peptide substrate. Below are general, literature-based workflows for kinase assays using FRATtide; optimize to your platform.

• Radiometric kinase assay (general):

1. Prepare 2× buffer (50 mM HEPES pH 7.5, 20 mM MgCl2, 2 mM DTT).
2. Combine equal volumes of enzyme mix (GSK‑3, γ‑32P‑ATP at 10–50 μM) and substrate mix (FRATtide at 10–200 μM).
3. Incubate 25–30°C for 10–30 min; quench with 3% phosphoric acid.
4. Spot on P81 filters, wash with 0.5% phosphoric acid, dry, and count CPM. • ADP‑Glo–type luminescent assay (general):
5. Set up reactions with FRATtide and ATP near Km in low‑phosphate buffer.
6. Incubate to desired conversion (<20%).
7. Add ADP‑Glo reagents per manufacturer instructions; read luminescence. • Phospho‑specific ELISA/dot‑blot (sequence permitting):
8. Immobilize FRATtide (e.g., biotinylated) on streptavidin plates.
9. Phosphorylate with GSK‑3; wash.
10. Detect with phospho‑motif antibody; develop colorimetric/fluorescent signal. • Data quality tips:

• Verify linearity with time and enzyme concentration.
• Include known inhibitor control and ATP titration to benchmark Z′ factor.

Item-specific validated protocols and recommended concentrations are not provided; refer to CoA/Spec Sheet and adapt to your detection chemistry.

Biological Roles

• Biological context (literature/general): FRATtide is derived from the human FRAT1 protein, an inhibitor/modulator of glycogen synthase kinase‑3 (GSK‑3). The peptide encompasses a GSK‑3 recognition motif, making it a convenient surrogate substrate in biochemical assays. • Kinase pathway relevance: GSK‑3α/β are Ser/Thr kinases implicated in diverse signaling cascades, including Wnt/β‑catenin, glycogen metabolism, and microtubule dynamics. GSK‑3 preferentially phosphorylates substrates primed by a prior phosphorylation event four residues C‑terminal to the target Ser/Thr, a feature recapitulated by FRATtide variants used in vitro. • Utility in systems biology:

• Enables quantification of GSK‑3 catalytic efficiency (kcat/Km) independent of full‑length protein substrates.
• Serves as a standardized readout to compare isoform selectivity (GSK‑3α vs GSK‑3β) and to benchmark small‑molecule inhibitors under controlled conditions. • Not a therapeutic or biological modifier in cells: FRATtide is intended for in vitro enzymology; it is not designed as a cell‑permeable probe or therapeutic agent. • Post‑translational modifications: In assays, phosphorylation occurs on the central serine/threonine of the FRATtide motif; the phosphorylated form can be detected by radiometric, MS, or phospho‑specific immunoassays (literature/general).

Note: Specific sequence, presence/absence of priming phosphate, and any terminal modifications for this item are not specified; refer to CoA/Spec Sheet.

Buffer Applications

FRATtide is used in enzyme assays rather than as a buffer component; nevertheless, buffer composition is critical for performance in kinase reactions.

• Typical kinase assay buffers (literature/general guidance):

• 20–50 mM HEPES or Tris, pH 7.0–7.5.
• 5–20 mM MgCl2 (essential cofactor for ATP binding/turnover).
• 0.01–0.1% Tween‑20 to reduce nonspecific adsorption in microplates if needed.
• 1–5 mM DTT or TCEP to maintain reducing conditions (verify compatibility with detection method).
• 0.1–1 mg/mL BSA can minimize peptide adsorption to plastics; confirm it does not interfere with detection. • Additives to avoid or control:
• High concentrations of phosphate or EDTA can inhibit kinase activity or coupled detection chemistries.
• Keep DMSO ≤1–2% v/v final unless validated otherwise. • Practical notes:
• Prepare fresh ATP solutions; store on ice during setup.
• Include no‑enzyme and no‑substrate controls to baseline the assay signal.

Item-specific buffer recipes and concentrations are not specified for this product; consult the CoA/Spec Sheet and adapt standard kinase assay buffers to your enzyme and detection platform.

Green Alternatives

As a peptide substrate, FRATtide itself is not readily replaced by a “greener chemical,” but greener practices can be applied to its use and associated solvents.

• Solvent and assay medium choices (general):

• Prefer aqueous buffers (HEPES, MOPS, PBS) over organic cosolvents; keep DMSO ≤1–2% v/v in final kinase reactions when possible to reduce solvent load and preserve enzyme activity.
• If organic aid is needed for dissolution, use minimal acetonitrile which is lower toxicity/viscosity than DMF and easier to remove; avoid halogenated solvents entirely. • Energy/resource efficiency:
• Conduct assays at ambient temperature when consistent with enzyme stability to reduce cooling/heating energy.
• Employ microplate (384/1536‑well) miniaturization to decrease reagent volumes and waste. • Waste minimization:
• Use ready‑to‑use buffers or concentrate formats to reduce packaging and solvent waste.
• Adopt no‑wash luminescent/fluorescent coupled assays where compatible to minimize aqueous waste streams. • Supply chain considerations:
• Batch larger aliquots upon first reconstitution to avoid multiple freeze–thaw cycles and product loss, improving overall efficiency.

Comparison (general):

• Aqueous buffers: greenest choice; excellent enzyme compatibility.
• DMSO: effective co‑solvent at low %, but aim to limit.
• DMF/MeCN: avoid DMF; prefer MeCN sparingly when necessary.

Item-specific solvent requirements are not specified; optimize empirically for your assay while following green chemistry principles.

Pharmaceutical Uses

This product is intended for research use only and is not an excipient or active ingredient.

• Role in pharmaceutical R&D (general):

• Tool substrate for high‑throughput screening and characterization of GSK‑3 inhibitors during lead discovery and optimization.
• Supports mechanistic enzymology, selectivity panels, and SAR by enabling consistent, standardized substrate conditions. • Regulatory status: No pharmacopeial monograph or excipient designation is indicated. Not for human or veterinary use. • Formulation considerations: Not applicable—FRATtide is not formulated for administration. When used in screening workflows, prepare assay‑ready solutions in validated, low‑binding labware to ensure reproducibility.

Item-specific regulatory designations or compliance statements are not provided; refer to CoA/Spec Sheet for any available quality documentation.

Physical Properties

Item-specific physicochemical specifications are not provided in the Product Data.

• Appearance: Not specified for this item; refer to CoA/Spec Sheet. • Molecular weight: Not specified for this item; refer to CoA/Spec Sheet. • Solubility (general, literature):

• Peptides of this class typically dissolve in aqueous buffers (e.g., water, PBS, HEPES) at 0.1–5 mg/mL; addition of 10–20% acetonitrile or small amounts of DMSO can aid dissolution for hydrophobic sequences.
• Acidifying to pH ~3 (0.1% TFA or acetic acid) or basifying slightly (≤pH 8) may improve solubility, depending on sequence. • pH stability (literature/general): Most unmodified peptides are stable in neutral to slightly acidic solutions; prolonged exposure to high pH can promote deamidation (Asn/Gln) and racemization. • Hygroscopicity (general): Lyophilized peptides can be mildly hygroscopic; handle quickly at ambient conditions to minimize moisture uptake. • Partitioning (general): Highly polar/hydrophilic; logP not typically defined for peptides. • Optical activity (general): Peptides of L‑amino acids are optically active; specific rotation is sequence‑dependent. • Thermal properties: Melting point and boiling point are not meaningful for lyophilized peptides; decomposition occurs before melting (literature/general).

For definitive, item-specific values (mass, purity, counter‑ion content, water content), consult the CoA/Spec Sheet.

Quality & Grades

• Grade/purity: Not specified for this item; refer to CoA/Spec Sheet. • Typical peptide QC (general information):

• Identity confirmation by ESI‑MS or MALDI‑TOF with expected [M+H]+ mass match.
• Purity by analytical RP‑HPLC (often reported as area % at 214/220 nm). Kinase substrate peptides are commonly supplied at ≥90–95% purity for enzymology; actual value for this item is not specified.
• Counter‑ions (e.g., TFA) and residual solvents may be present in lyophilizates; quantitative levels for this item are not specified. • Implications for use:
• Higher HPLC purity reduces background signal in kinase assays and improves lot‑to‑lot consistency.
• Salt form and counter‑ion content can slightly affect solubility and mass calculations; confirm with CoA for precise molarity preparations. • Documentation: For this catalog item, authoritative specifications (purity %, sequence confirmation, water content, residual solvent, endotoxin/bioburden if applicable) are provided on the CoA/Spec Sheet. Use those values for regulated or quantitative workflows.

Reaction & Applications

This product is a peptide substrate optimized for kinase biochemistry rather than synthetic organic reactions.

• Core application (literature/general):

• Substrate for glycogen synthase kinase‑3 (GSK‑3α/β) activity assays. FRATtide contains a serine‑centered phosphoacceptor motif embedded in a proline/arginine‑rich context that satisfies GSK‑3’s preference for a pre‑phosphorylated priming site at +4 (depending on sequence variant) and consensus S/T‑X‑X‑X‑S(p) pattern. • Assay modalities:
• Radiometric kinase assays (γ‑32P‑ATP incorporation onto FRATtide; filter‑binding or scintillation proximity formats).
• ADP‑Glo/ADP‑Hunter type coupled assays measuring ADP formation in presence of FRATtide.
• Antibody‑based detection using phospho‑specific antibodies to the phosphorylated FRATtide motif (ELISA or dot‑blot), sequence permitting.
• LC‑MS or CE quantitation of phosphorylated vs unphosphorylated peptide. • Use in inhibitor profiling:
• Benchmark peptide for screening/IC50 determination of GSK‑3 inhibitors under defined ATP/substrate concentrations; supports kinetic parameterization (Km, Vmax) of GSK‑3 with a standardized substrate. • Experimental considerations:
• Magnesium dependence (typically 5–20 mM Mg2+) and ATP concentration near Km for ATP minimize off‑target effects (literature/general).
• Include controls lacking enzyme, and a primed peptide if your FRATtide variant requires priming.
• Avoid high phosphate in buffers for certain coupled assays; HEPES or Tris are commonly used. Item-specific sequence and kinetic constants are not specified; refer to CoA/Spec Sheet and primary literature for exact parameters.

Reaction Conditions

FRATtide is used in enzymatic phosphorylation reactions rather than chemical transformations. The following represent general kinase assay conditions from literature; adjust empirically.

• Typical GSK‑3 assay setup (literature/general):

• Buffer: 25–50 mM HEPES, pH 7.0–7.5; 10 mM MgCl2; 1 mM DTT.
• Substrate (FRATtide): 5–200 μM depending on Km and detection window.
• ATP: 10–100 μM near the kinase’s Km for ATP; increase to saturating concentrations for Vmax determination.
• Enzyme: 0.1–50 nM recombinant GSK‑3α/β, adjusted to maintain initial‑rate conditions with <10–20% substrate consumption.
• Temperature/time: 25–30°C for 10–60 min; verify linearity over time. • Detection options:
• Radiometric incorporation using γ‑32P‑ATP with filter binding or SPA beads.
• Luminescent ADP detection (e.g., ADP‑Glo) with appropriate quench/convert steps; avoid phosphate‑rich buffers.
• LC‑MS endpoint quantitation for phosphorylated peptide. • Controls:
• No‑enzyme, no‑substrate, and known GSK‑3 inhibitor controls (e.g., CHIR99021) to verify assay performance. • Interferences:
• High salt and detergents can affect enzyme activity or detection; validate matrix effects. Keep DMSO low.

Item-specific kinetic constants (Km, kcat) and validated concentration ranges are not specified; refer to CoA/Spec Sheet and primary references for exact parameters.

Safety & Handling

• GHS classification, signal word, pictograms, H‑statements: Not specified for this item; refer to SDS. • General hazards (peptides): Typically considered low hazard in research use; avoid inhalation of powders and contact with eyes/skin. Handle in a clean area to prevent contamination (literature/general). • PPE: Lab coat, safety glasses, and appropriate disposable gloves. Use a dust mask or work in a fume hood/biological safety cabinet when weighing lyophilized material to avoid aerosolization (general best practice). • First aid (general guidance; defer to SDS):

• Inhalation: Move to fresh air; seek medical advice if symptoms persist.
• Skin/eye contact: Rinse with water for several minutes; remove contaminated clothing.
• Ingestion: Rinse mouth; seek medical attention if unwell. • Handling notes:
• Minimize repeated freeze–thaw cycles; prepare single‑use aliquots upon first reconstitution.
• Use sterile technique if employing in cell‑free enzymology that demands low bioburden. • Incompatibilities (general): Strong oxidizers and extreme pH can degrade peptide side chains; avoid prolonged exposure to elevated temperatures. Metal contamination is not typically critical for kinase assays but chelators (e.g., EDTA) may interfere with Mg2+/ATP‑dependent reactions. • Waste disposal: Dispose according to institutional and local regulations for non‑hazardous laboratory chemicals unless SDS indicates otherwise. Always consult the product SDS for authoritative safety and regulatory information.

Solvent Selection

FRATtide is a hydrophilic peptide substrate; solvent choice focuses on complete dissolution without altering kinase activity.

• Primary solvents (general/literature):

• Water or aqueous buffers (e.g., 1× PBS, 20–50 mM HEPES, pH 7.0–7.5) are preferred for immediate assay use.
• For initial stock solutions, nuclease‑/protease‑free water with 0.1% TFA or acetic acid can aid dissolution of basic peptides; alternatively, 10–20% acetonitrile or ≤10% DMSO may be used, verifying compatibility with downstream assays. • Miscibility/compatibility:
• Fully miscible with water; limited solubility expected in purely nonpolar solvents.
• High DMSO (>2–5% v/v final) can inhibit certain kinases or alter Km values; keep final organic cosolvent low and include appropriate controls. • Practical tips:
• Warm gently to room temperature and vortex/sonicate briefly to dissolve; avoid strong base which can induce deamidation.
• Filter sterilize (0.22 μm) only if necessary; adsorption losses to filters may occur—pre‑rinse with buffer containing 0.1% BSA if adsorption is problematic (test first). • Comparison (general):
• Water/buffer: maximal biocompatibility; best for immediate kinase assays.
• DMSO stocks: useful for poorly soluble sequences; ensure low final DMSO in enzyme reactions.
• Acetonitrile/water: aids dissolution but may perturb proteins above a few percent. Always validate solvent effects on your specific assay readout.

Storage & Reconstitution

• Storage conditions (from Product Data): Store at -20°C. • Shipping (from Product Data): Shipped in an ice chest with ice pads to maintain low temperature. • Long-term handling (general best practice for peptides):

• Keep lyophilized material desiccated, protected from light, and tightly capped. For storage beyond several months, −20°C or below is recommended; for multi‑year storage, −80°C is preferred.
• After first opening, allow vial to equilibrate to room temperature in a desiccator before uncapping to minimize moisture condensation. • Reconstitution (general guidance):
• Use sterile, nuclease/protease‑free water or assay buffer to prepare a concentrated stock (e.g., 1–5 mM or as solubility allows). If dissolution is slow, add up to 10–20% acetonitrile or small amounts of DMSO; confirm assay compatibility.
• Vortex gently and, if needed, brief bath sonication. Avoid strong base; adjust pH modestly if required. • Aliquoting and stability:
• Prepare single‑use aliquots; avoid repeated freeze–thaw cycles. Store aliquots at −20°C or −80°C. Thaw on ice and keep cold during setup.
• Working solutions are typically kept at 2–8°C for the assay day; for longer periods, flash‑freeze and store at −80°C. • Specifications such as exact sequence, salt form, counter‑ion content, and peptide concentration after reconstitution are not specified for this item; refer to CoA/Spec Sheet.

Structure & Identity

• Item: FRATtide (SKU: F1448880); a synthetic peptide substrate derived from the FRAT1 (Frequently Rearranged in Advanced T‑cell lymphomas) protein region used for GSK‑3 kinase assays. • CAS: 251087-38-4 • Molecular formula: Not specified for this item; refer to CoA/Spec Sheet. • Molecular weight: Not specified for this item; refer to CoA/Spec Sheet. • SMILES: Not specified for this item; refer to CoA/Spec Sheet. • InChIKey: Not specified for this item; refer to CoA/Spec Sheet. • Structural features (general):

• Peptidic backbone composed of L‑α‑amino acids with amide bonds; contains side chains typical of serine-, proline-, arginine‑rich motifs that enable recognition by glycogen synthase kinase‑3 (GSK‑3) (literature/general).
• Often supplied as the non‑phosphorylated substrate peptide; may carry free N‑terminus/C‑terminus unless otherwise modified (literature/general). Specific terminal modifications for this item are not specified; refer to CoA/Spec Sheet. • 2D structural description (general): A linear sequence of amino acid residues with alternating carbonyl‑amide linkages; side chains project alternately along the backbone. Serine/threonine residues present potential phosphorylation sites; clustered basic residues (e.g., arginine/lysine) enhance kinase interaction (literature/general). • Identity confirmation (typical for peptides): LC‑MS and analytical RP‑HPLC are commonly used for identity/purity assessment; exact methods/acceptance criteria for this item are not specified; refer to CoA/Spec Sheet.

Synthetic Utility

Although not a building block for small‑molecule synthesis, FRATtide can be a versatile component in biochemical probe development and peptide chemistry workflows.

• Peptide modification (literature/general):

• Terminal functionalization (biotinylation, fluorescent dyes, affinity tags) enables capture/detection in ELISA, TR‑FRET, or FP assays. Site‑selective labeling can be performed on N‑terminus, Lys ε‑amines, or via introduced handles (Azido/Alkyne) using CuAAC.
• Phosphorylated variants (primed FRATtide) can be synthesized to probe GSK‑3 processivity and priming dependence. • Solid‑phase peptide synthesis (SPPS):
• Standard Fmoc/tBu chemistry on Rink amide or Wang resin is used to assemble FRATtide sequences; difficult motifs may benefit from pseudoproline dipeptides or optimized coupling reagents (e.g., HATU/DIC‑Oxyma) to improve yields. • Assay conjugates:
• Immobilization on beads/plates through biotin–streptavidin or maleimide–thiol chemistry supports pull‑down or enzyme kinetic formats. • Retrosynthetic value: As a defined epitope‑like substrate, FRATtide allows modular swapping of residues to map kinase specificity and generate SAR around GSK‑3 recognition motifs.

Item-specific sequence, modifications, and resin history are not specified; consult the CoA/Spec Sheet if such details are required for method development.

Target Specificity

Target specificity fields (antigen, clone, isotype, species reactivity) do not apply to this product, which is a peptide substrate rather than an antibody or affinity reagent.

• Functional target (general): Serves as a substrate for glycogen synthase kinase‑3 (GSK‑3α/β) in biochemical assays. • No antibody clone/isotype or species specificity is associated with this item.

For assay design, validate that your kinase preparation (isoform and source) efficiently phosphorylates the specific FRATtide sequence provided with your lot; item-specific sequence information is not specified here—refer to CoA/Spec Sheet.