⚠️ FOR IN-VITRO LABORATORY RESEARCH USE ONLY This laboratory reference is prepared exclusively for scientific research and analytical reference. TB-500 (Thymosin Beta-4 synthetic analog) is an experimental reagent designated strictly for in-vitro laboratory experimentation. It is not approved for human or veterinary administration, clinical therapy, or diagnostic use. No medical advice or dosing guidelines are provided.
Chemical Profile & Molecular Characterization
TB-500 is the synthetic analog of the endogenous polypeptide Thymosin Beta-4 (Tβ4). While native Thymosin Beta-4 has a calculated molecular weight of 4,921 Da, synthetic TB-500 reference standards incorporate an N-terminal acetyl cap, yielding a molecular weight of approximately 4,963 Da. Thymosin Beta-4 is naturally occurring and found in all eukaryotic cell types, with highest concentrations in blood platelets, leukocytes, and wound exudates.
The peptide acts primarily as the cell’s dominant G-actin sequestering molecule. Within its 43-amino acid sequence, the key active site is the central heptapeptide motif LKKTETQ (residues 17–23). This domain binds monomeric G-actin in a 1:1 complex, inhibiting assembly into F-actin while maintaining an actin monomer pool for rapid cellular remodeling. In preclinical laboratory models, TB-500 is investigated for its role in stimulating cell migration, accelerating wound healing, promoting angiogenesis, downregulating NF-κB, and suppressing pro-inflammatory cytokines (TNF-α, IL-6).
| Parameter | Value / Profile |
|---|---|
| Compound Name | TB-500 (Thymosin Beta-4 Synthetic Analog) |
| Parent Polypeptide | Thymosin Beta-4 |
| CAS Registry Number | 77591-33-4 (Thymosin Beta-4) |
| Molecular Weight | ~4,963 Da (TB-500) / 4,921 Da (native Tβ4) |
| Sequence | Ac-SDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES |
| Active Domain | LKKTETQ (Residues 17–23) |
| Compound Category | Thymosin Peptides |
| Physical Form | Sterile white lyophilized powder |
| Solubility | Soluble in water, BAC water, and PBS (>10 mg/mL) |
Reconstitution Protocol & Diluent Dynamics
Lyophilized TB-500 dissolves readily due to its charged, hydrophilic residues. However, careful handling protects against mechanical shear stress and foaming.
Diluent Selection
- Bacteriostatic Water (0.9% Benzyl Alcohol): Preferred for multi-use research workflows; inhibits microbial proliferation at 2–8°C for up to 28 days.
- Sterile Water / Saline (0.9% NaCl): Recommended for cell culture assays sensitive to benzyl alcohol cytotoxicity. Use within 24 hours or freeze in single-use aliquots.
Reconstitution Table
| Vial Mass | Diluent Volume | Final Concentration | Draw per 100 mcg |
|---|---|---|---|
| 2 mg | 1.0 mL | 2.0 mg/mL (2,000 mcg/mL) | 0.05 mL |
| 2 mg | 2.0 mL | 1.0 mg/mL (1,000 mcg/mL) | 0.10 mL |
| 5 mg | 2.0 mL | 2.5 mg/mL (2,500 mcg/mL) | 0.04 mL |
| 5 mg | 2.5 mL | 2.0 mg/mL (2,000 mcg/mL) | 0.05 mL |
| 10 mg | 2.0 mL | 5.0 mg/mL (5,000 mcg/mL) | 0.02 mL |
| 10 mg | 5.0 mL | 2.0 mg/mL (2,000 mcg/mL) | 0.05 mL |
Reconstitution Steps
- Equilibrate the lyophilized vial and diluent to room temperature (20–25°C) for 15–20 minutes.
- Disinfect the septum with 70% isopropanol and allow to dry.
- Angle the needle toward the inner glass wall so diluent flows down slowly without spraying the cake.
- Gently swirl in circular motions. Do not shake or vortex, as turbulent foaming induces aggregation.
- Confirm complete solution clarity within 2–5 minutes.
Lyophilized Storage & Temperature Stability
Under vacuum or inert gas, lyophilized TB-500 exhibits robust chemical stability. Primary degradation pathways include methionine oxidation at Met6 (+16 Da mass shift), deamidation of asparagine (Asn26) and glutamines, and slow peptide cleavage.
Lyophilized Stability Matrix
| Temperature | Shelf Life | Purity | Storage Protocol & Notes |
|---|---|---|---|
| -80°C (Ultra-Low) | 36–48 months | >98% | Primary archival storage with desiccant in darkness. |
| -20°C (Standard) | 24 months | >98% | Routine research inventory. Avoid auto-defrost cycling. |
| 2°C to 8°C (Refrigerated) | 12–18 months | >95% | Active-use stock; keep sealed against moisture. |
| 20°C to 25°C (Ambient) | 30–60 days | >92% | Tolerable during transit or benchtop handling. |
| >37°C (Thermal Stress) | <72 hours | <85% | Accelerated deamidation and peptide bond hydrolysis. |
Reconstituted Stability & Environmental Sensitivities
Once dissolved, the protective dry matrix is removed, accelerating hydrolysis and oxidation.
Reconstituted Solution Stability
| Condition | Diluent | Window | Primary Degradation Risk |
|---|---|---|---|
| 2°C to 8°C | Bacteriostatic Water | 21–28 days | Slow Met6 oxidation, minor deamidation |
| 2°C to 8°C | Sterile Water / Saline | 24–48 hours | Rapid bacterial growth, proteolysis |
| -20°C | Sterile Diluent | 60–90 days | Ice crystal shearing; single freeze only |
| -80°C | Sterile Water / PBS | 6–12 months | Optimal preservation for stock solutions |
| 20°C to 25°C | Any Diluent | <8 hours | Hydrolysis and microbial proliferation |
Light Sensitivity & Photodegradation
TB-500 is light-sensitive due to photo-labile residues (Phe12, Met6) vulnerable to photo-oxidation under UV and short-wavelength visible light. Photo-excitation generates singlet oxygen, accelerating methionine sulfoxide conversion and covalent dimerization. Vials must be stored in amber glass or wrapped in aluminum foil.
Freeze-Thaw Impact & Aliquoting Protocols
Repeated freeze-thaw cycles degrade reconstituted TB-500:
- Cryoconcentration: Advancing ice fronts concentrate solute and peptide molecules into liquid microdomains, driving aggregation.
- Interfacial Denaturation: Ice-water boundary shear forces unfold peptide tertiary structure, causing irreversible precipitation.
Laboratory Recommendation: Never subject reconstituted TB-500 to repeated freeze-thaw cycles. Divide freshly reconstituted peptide into single-use aliquots (50–200 µL) in low-binding polypropylene tubes and freeze at -20°C or -80°C. Thaw each aliquot once immediately before testing.
Analytical Quality Testing & Purity Verification
Rigorous quality testing is essential to confirm peptide integrity and purity before experimental use.
Reverse-Phase HPLC (RP-HPLC)
RP-HPLC assesses chromatographic purity and separates degradation impurities:
- Column: C18 reverse-phase silica column (4.6 × 250 mm, 5 µm, 300 Å pore size).
- Mobile Phases: Buffer A (0.1% TFA in H₂O) and Buffer B (0.1% TFA in acetonitrile).
- Elution: Linear gradient of 10% to 50% Buffer B over 30 minutes at 1.0 mL/min.
- UV Detection: Dual wavelengths at 214 nm (peptide backbone) and 280 nm.
- Specification: Main peak area ≥ 98.0%. Symmetrical peak shape; satellite peaks indicate deamidated or oxidized forms.
Mass Spectrometry (MS)
- ESI-MS / MALDI-TOF: Confirms molecular weight (~4,963.5 Da for TB-500; 4,921.3 Da for native Tβ4).
- Charge States: ESI-MS reveals multiply charged ions ([M+3H]³⁺, [M+4H]⁴⁺, [M+5H]⁵⁺).
- Degradation Detection: Identifies +16 Da shifts (methionine oxidation), +1 Da shifts (deamidation), and synthesis truncations.
Additional Quality Controls
- Peptide Content: Amino acid analysis / UV spectroscopy confirms net peptide content (typically 75–85% peptide, balance counterions and water).
- Endotoxin Testing (LAL Assay): Confirms endotoxins ≤ 0.05 EU/mg to prevent inflammatory artifacts in cell assays.
Standard Laboratory Storage & Handling Protocol
- Receipt & Inspection: Verify cold-chain pack integrity, inspect the lyophilized cake, and archive manufacturer Certificate of Analysis (CoA) records.
- Lyophilized Storage: Store vials sealed at -20°C or -80°C with desiccant, protected from ambient light.
- Reconstitution: Equilibrate vial to 20–25°C for 20 minutes. Introduce diluent down the glass wall under aseptic laminar hood conditions. Swirl gently without shaking.
- Aliquot Architecture: Partition freshly reconstituted solution into single-use low-retention tubes for freezing (-20°C or -80°C) to prevent repeated freeze-thaw cycles.
- Quality Control: Periodically verify solution clarity and run RP-HPLC/MS identity confirmation prior to high-sensitivity in-vitro assays.