Actin (G-actin). Thymosin β4 (human, bovine, horse, rat) (CAS#: 77591-33-4) binds monomeric actin with a dissociation constant (Kd) of 0.7–1 μM for rabbit skeletal muscle actin [1]; Kd values between 0.5 and 2.5 μM are reported for various β-thymosins [2]; the sulfoxide form (oxidation at Met6) has ~20-fold higher Kd [2].

Thymosin β4 (human, bovine, horse, rat) (CAS#: 77591-33-4) (recombinant, 44 residues with initiator Met not cleaved) expressed in E. coli and purified by heat treatment (80°C, 10 min) and ion-exchange chromatography (Mono Q, Mono S) retains actin-binding activity. Equilibrium sedimentation shows it is primarily monomeric (91% monomer) with a molecular weight of 5,889 Da (calculated 5,057 Da). Gel filtration suggests an apparent molecular mass of ~20 kDa due to extended conformation. In non-denaturing gels, Tβ4 migrates slower than actin and forms a complex with actin that migrates ahead of actin. Tβ4 inhibits actin polymerization in a dose-dependent manner: with 2.3 μM Tβ4, the apparent critical concentration of pyrene-labeled actin increases from 0.2 μM to 0.53 μM, yielding a Kd of 1.19 μM. Pyrene-actin fluorescence assays show that Tβ4 at 0.77–3.08 μM decreases the rate of barbed end elongation; the observed rates are slightly higher than predicted from steady-state Kd, especially at low Tβ4/actin ratios. Tβ4 does not bind polyphosphoinositides (PIP2 up to 128 μM) and its activity is unaffected by PIP2. At high concentrations (>100 μM), Tβ4 can be cross-linked to F-actin and incorporated into filaments. Oxidation of Met6 to sulfoxide (H2O2 treatment) increases Kd ~20-fold and reduces actin-binding affinity. N-terminal truncation (removing residues 1-6 or 1-12) also increases Kd, while truncation of residues 1-23 or 26-43 abolishes binding. Tβ4 is a glutaminyl substrate for transglutaminase, allowing cross-linking to extracellular matrix proteins. [1][2]
In HUVECs, Thymosin β4 (human, bovine, horse, rat) (CAS#: 77591-33-4) (0.1 μg/ml) promotes attachment, spreading, and tube formation on Matrigel. In coronary artery ring assays, Tβ4 (0.1 μg/ml) induces capillary sprouting and increases vessel area (doubling of vessel area via branching). Tβ4 acts as a chemoattractant for endothelial cells (HUVEC and human coronary artery endothelial cells) in Boyden chamber assays (4- to 6-fold over media alone), but does not attract fibroblasts, smooth muscle cells, neutrophils, monocytes, or HT1080 cells. The actin-binding motif LKKTET is necessary and sufficient for angiogenic activity. Tβ4 up-regulates VEGF expression in B16-F10 lung tumor cells and in the developing heart; knockdown of Tβ4 reduces VEGF expression. Tβ4 also increases expression of cytoskeletal proteins (myosin IIA, α-actinin, tropomyosin, talin, α5-integrin, vinculin, zyxin), ECM components (laminin-5), and matrix metalloproteinases (MMP-2, MMP-9, MT6-MMP). In epicardial explant cultures from adult mouse hearts, Tβ4 stimulates outgrowth of epicardium-derived cells (EPDCs) that differentiate into smooth muscle (SMαA-positive) and endothelial (Tie2-positive) cells. Addition of Tβ4 significantly increases numbers of SMαA- and Tie2-positive cells; this effect is further enhanced by VEGF and FGF7. Tβ4 is detected in the nucleus, suggesting possible transcriptional regulatory functions. [2][3]

In vivo, endogenous Thymosin β4 (human, bovine, horse, rat) (CAS#: 77591-33-4) is required for coronary vasculogenesis, angiogenesis, and arteriogenesis during mouse embryonic development. Conditional myocardial knockdown of Tβ4 (using Tβ4shRNA driven by Nkx2.5Cre) results in a thin non-compacted myocardium, detached epicardium with surface nodules, defective coronary vessels (Tie2-positive microvessels absent), lack of smooth muscle cell recruitment (SMαA-positive cells trapped in epicardium), hemorrhaging from large thoracic vessels, and absence of the right subclavian artery. Tβ4 knockdown embryos also show reduced myocardial AcSDKP levels (40% reduction). Injection of pregnant females with AcSDKP restores tetrapeptide levels but does not rescue the phenotype. Following myocardial infarction in adult mice, endogenous Tβ4 and AcSDKP levels are up-regulated in the heart. Topical or intraperitoneal administration of Tβ4 accelerates wound healing in rat full-thickness wound models (increased epithelialization, collagen deposition, angiogenesis, and wound contraction). In aged and diabetic rodents, Tβ4 also promotes wound repair. In corneal injury models, topical Tβ4 promotes corneal epithelial cell migration, increases cell-cell and cell-matrix contacts, inhibits apoptosis, suppresses MMP-2/MMP-9/MT6-MMP, and reduces inflammation via NFκB/TNF-α pathway. In chick chorioallantoic membrane (CAM) assays, Tβ4 enhances angiogenesis (doubling of vessel area) comparable to positive control β-PMA. Tβ4 overexpression in B16-F10 cells increases tumor angiogenesis and VEGF expression. [2][3]

Equilibrium sedimentation: Thymosin β4 (human, bovine, horse, rat) (CAS#: 77591-33-4) (11.5 μM in 20 mM Tris-HCl, pH 7.4) is centrifuged at 40,000 rpm at 277 K in an An60-Ti rotor. Absorbance scans at 220 nm are taken at equilibrium; molecular weight is calculated using a partial specific volume of 0.7149 cm³/g, yielding a buoyant molecular weight of 1,608 and a calculated molecular weight of 5,889 Da, indicating ~91% monomer. [1]
Pyrene-actin polymerization assay: Rabbit muscle actin labeled with N-(1-pyrenyl)iodoacetamide (1:10 or 1:6.7 ratio) is mixed with unlabeled actin in G-buffer (0.5 mM ATP, 0.5 mM β-mercaptoethanol, 0.2 mM KCl, 10 mM Tris-HCl, pH 7.5). Polymerization is initiated by adding F-buffer (0.1 M KCl, 1.3 mM MgCl2, 0.5 mM ATP, 0.5 mM β-mercaptoethanol, 0.2 mM CaCl2, 10 mM Tris-HCl, pH 7.5). Fluorescence is measured at 12 and 24 h. For barbed end elongation assays, preformed gCap39-capped actin seeds are added to Mg2+-actin (1.5 μM) in the presence of Tβ4 (0–3.08 μM); polymerization is initiated by adding salt and 5 mM EGTA, and the initial linear slope is recorded. [1]
Non-denaturing gel electrophoresis: 7.5% acrylamide, 0.2% bis gels containing 25 mM Tris, 0.4 M glycine, 0.2 mM ATP are used. Tβ4 (0.6–1.2 μg) is mixed with rabbit muscle actin monomers (2.2 μg) in G-buffer, incubated 10 min at room temperature, then glycerol with bromphenol blue is added, and electrophoresis is performed at 10°C, 20–25 V/cm. [1]
Actin sedimentation assay: 2.6 μM actin (0–100% pyrene-labeled) is incubated with 0–4 μM Tβ4 in F-buffer for 24 h at room temperature, then centrifuged at 20 psi in an airfuge for 30 min. Pellets and supernatants are analyzed by SDS-PAGE. [1]
UV absorption spectrum: Tβ4 concentration is determined by amino acid analysis; extinction coefficient at 205 nm is 350 mM⁻¹ cm⁻¹. [1]
Cross-linking: Zero-length cross-linker EDC (1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide) is used to map interaction sites: Lys3 of Tβ4 cross-links to Glu167 of actin; Lys18 cross-links to one of the N-terminal acidic residues (DEDE); Lys38 cross-links to Gln41 of actin via transglutaminase. [2]
DNase I inhibition assay: G-actin binding is assessed by measuring inhibition of DNase I activity. [2]
Ultrafiltration assay for Kd determination: Mixtures of Tβ4 and G-actin are incubated and free actin is separated by ultrafiltration; bound complex is quantitated. [2]
Epicardial explant culture: Adult mouse hearts are dissected, epicardium is peeled, cut into small pieces, and placed on Matrigel-coated coverslips in culture medium. Tβ4 is added, and outgrowth of epicardium-derived cells is observed after 3–5 days. Differentiated cells are identified by immunostaining for SMαA (smooth muscle), Tie2 (endothelial), and procollagen type I (fibroblasts). [3]

HL60 cell differentiation: DMSO-treated HL60 cells show increased Thymosin β4 (human, bovine, horse, rat) (CAS#: 77591-33-4) mRNA levels along with β-actin mRNA. [2]
PC12 cells: Nerve growth factor (NGF) or fibroblast growth factor (FGF) increases Tβ4 mRNA tenfold. [2]
Rat thymocyte stimulation: Concanavalin A induces a transient sixfold increase in Tβ4 peptide within 1 hour without elevation of mRNA; during S-phase, a 2.5-fold increase in Tβ4 is accompanied by corresponding mRNA increase. [2]
NIH 3T3 cells: Overexpression of thymosin β10 (but not Tβ4) results in thicker actin filaments, increased cell motility, faster spreading, and higher chemotactic and wound healing activity. Stable overexpression of Tβ4 leads to increased G-actin and F-actin, unchanged G/F ratio, and increased levels of cytoskeleton-related proteins (myosin IIA, α-actinin, tropomyosin, talin, α5-integrin, vinculin) without changing profilin or ADF. [2]
HUVEC migration assay: Boyden chambers with polycarbonate membranes (8 μm pores) are coated with collagen; HUVECs are placed in the upper chamber, and Tβ4 (1–100 nM) in the lower chamber acts as a chemoattractant. After 4–6 h, migrated cells on the lower membrane surface are stained and counted. Tβ4 stimulates directional migration 4- to 6-fold over control. [3]
Coronary artery ring assay: Coronary arteries are isolated from adult rat hearts, cut into 1–2 mm rings, embedded in Matrigel, and cultured in medium with or without Tβ4 (0.1 μg/ml). Vessel sprouting is quantified after 7–10 days. [3]
Epicardial explant culture (detailed in Enzyme Assay): Outgrowth cells are characterized by immunocytochemistry using antibodies against SMαA, Tie2, Flk1, and procollagen type I. [3]
Cell viability/proliferation: Not directly assessed in these references; however, Tβ4 stimulates endothelial cell proliferation and differentiation on Matrigel. [3]

Transgenic mouse generation: A conditional Tβ4 knockdown mouse line is generated using a Tβ4-specific short hairpin RNA (shRNA) under control of a floxed transcriptional terminator. These mice are crossed with Nkx2.5Cre transgenic mice to achieve myocardial-specific deletion. Pregnant females are injected intraperitoneally with AcSDKP (no specific dose given) to rescue the knockdown phenotype. Embryos are harvested at E14.5, fixed, paraffin-embedded, sectioned, and stained with hematoxylin/eosin or subjected to immunohistochemistry with antibodies against Tie2, SMαA, and other markers. [3]
Rat full-thickness wound healing model: A 6 mm full-thickness skin wound is created on the dorsum of rats. Thymosin β4 (human, bovine, horse, rat) (CAS#: 77591-33-4) is administered either topically (in a gel or solution) or intraperitoneally (dose not specified in reviewed text). Wound closure is measured over 7–10 days; histological analysis assesses epithelialization, collagen deposition, and angiogenesis. [2]
Corneal injury model: Mouse cornea is abraded (alkali burn or mechanical debridement), and Tβ4 is applied topically (concentration not specified). Corneal epithelial healing is monitored by fluorescein staining; inflammatory mediators (NFκB, TNF-α) are assessed by immunoblotting or ELISA. [2]
Chick chorioallantoic membrane (CAM) assay: Fertilized chicken eggs are incubated, a window is opened in the shell, and a filter disc containing Thymosin β4 (human, bovine, horse, rat) (CAS#: 77591-33-4) (dose not specified) is placed on the CAM. After 48–72 h, angiogenesis is quantified by counting vessel branches or measuring vessel area. [2]
Myocardial infarction model (referenced but not performed in these papers): Coronary artery ligation in adult mice; Tβ4 treatment (dose not specified) improves cardiac function and reduces scar volume via Akt activation. [3]

Yuet al(1993) Thymosin u03b210 and thymosin u03b24 are both actin monomer sequestering proteins. J.Biol.Chem. 268502 PMID:8416954

Huffet al(2001) u03b2-thymosins, small acidic peptides with multiple functions. Int.J.Biochem.Cell Biol. 33205 PMID:11311852

Smartet al(2007) Thymosin u03b24 and angiogenesis: modes of action and therapeutic potential. Angiogenesis 10229 PMID:17632766

Thymosin β4 (human, bovine, horse, rat) (CAS#: 77591-33-4) is an acidic peptide (pI 4.7–5.0) with 43–44 amino acid residues. It is expressed ubiquitously in mammalian tissues, with high concentrations in platelets (200–500 μM), polymorphonuclear leukocytes (269–564 fg/cell), and mononuclear leukocytes (183–380 fg/cell); it is undetectable in erythrocytes. The peptide is largely unstructured in water but forms α-helical structures (residues 4–16 and 30–40) in fluorinated alcohols. It is not cleaved from a signal peptide; however, it can be secreted and detected in blood plasma (0.026–0.387 mg/L) and wound fluid (13 μM). The N-terminal tetrapeptide AcSDKP (N-acetyl-seryl-aspartyl-lysyl-proline) is generated by prolyl oligopeptidase or AspN-like protease cleavage and is a potent inhibitor of hematopoietic stem cell proliferation and an inducer of angiogenesis. AcSDKP is degraded exclusively by angiotensin-converting enzyme (ACE). Tβ4 sulfoxide (oxidized at Met6) has reduced actin-binding affinity but exhibits anti-inflammatory activity by inhibiting neutrophil migration and dispersion. The actin-binding site is localized to motif ¹⁷LKKTETQEK²⁵; the LKKTET sequence is both necessary and sufficient for actin binding and angiogenic activity. Tβ4 binds G-actin in an extended conformation, contacting subdomains 1, 2, and 3 of actin (cross-links: K3 to E167, K18 to DEDE, K38 to Q41). Tβ4 inhibits nucleotide exchange on G-actin (unlike profilin which accelerates it). The peptide is a substrate for transglutaminase (cross-linking to fibrin, collagen). Overexpression of Tβ4 is associated with increased metastatic potential in various cancers (colorectal, melanoma, breast, prostate, thyroid). Tβ4 is being evaluated in phase 2 clinical trials for epidermolysis bullosa and pressure ulcers. [1][2][3]

C212H350N56O78S

4963.44085168839

77591-33-4

16132341

Typically exists as solid at room temperature

0

72

88

180

347

12200

47

S(C)CC[C@@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@@H](CO)C(N[C@@H](CCCCN)C(N[C@@H](CC(C)C)C(N[C@@H](CCCCN)C(N[C@@H](CCCCN)C(N[C@@H]([C@@H](C)O)C(N[C@@H](CCC(=O)O)C(N[C@@H]([C@@H](C)O)C(N[C@@H](CCC(N)=O)C(N[C@@H](CCC(=O)O)C(N[C@@H](CCCCN)C(N[C@@H](CC(N)=O)C(N1CCC[C@H]1C(N[C@@H](CC(C)C)C(N1CCC[C@H]1C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(NCC(N[C@H](C(N[C@H](C(=O)O)CO)=O)CCC(=O)O)=O)=O)C)=O)CCC(N)=O)=O)CCCCN)=O)CCC(=O)O)=O)CCC(N)=O)=O)CCC(=O)O)=O)[C@@H](C)CC)=O)[C@@H](C)O)=O)CCC(=O)O)=O)CCCCN)=O)CO)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)CCCCN)=O)CC(=O)O)=O)CC1C=CC=CC=1)=O)CCCCN)=O)CCC(=O)O)=O)[C@@H](C)CC)=O)CCC(=O)O)=O)C)=O)NC([C@H](CC(=O)O)NC([C@@H]1CCCN1C([C@H](CCCCN)NC([C@H](CC(=O)O)NC([C@H](CO)NC(C)=O)=O)=O)=O)=O)=O

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)