TB-500 is a synthetic peptide fragment of thymosin β4 (Tβ4), the 43-residue protein that holds most of the unpolymerised actin in a cell. The fragment is the N-acetylated heptapeptide Ac-Leu-Lys-Lys-Thr-Glu-Thr-Gln, which carries the central actin-binding motif of the parent protein, and it is used in actin polymerisation, G-actin binding and cell migration assays. TB-500 from NuVion is supplied as a laboratory chemical for in vitro research use only.
Key facts
| Type | Synthetic peptide, thymosin β4 fragment (residues 17 to 23) |
| Amino acid count | 7 |
| Sequence | Ac-Leu-Lys-Lys-Thr-Glu-Thr-Gln (Ac-LKKTETQ) |
| Molecular formula | C38H68N10O14 |
| Molecular weight | 889.0 g/mol |
| CAS number | 885340-08-9 |
| Synonyms | Tβ4(17-23), thymosin β4 actin-binding fragment, TB500 |
| Supplied form | Lyophilised powder in a sealed vial |
Structure and chemistry
Thymosin β4 is a 43-residue, N-acetylated, intrinsically disordered protein with a mass of about 4.96 kDa. Its central segment, LKKTET at residues 17 to 22, is the actin-binding motif shared by the β-thymosin family and by the WH2 domains of actin regulators such as WASP, N-WASP and ciboulot. TB-500 is that motif plus the following glutamine, with an acetyl group on the N-terminus that mirrors the acetylated N-terminus of the parent protein and removes one positive charge. The name TB-500 is also used loosely for full-length Tβ4, so the mass on the Certificate of Analysis is the way to confirm which entity is in the vial: about 889 Da for the heptapeptide against about 4,960 Da for the protein.
The two lysines give plus 2, the glutamate and the C-terminal carboxylate give minus 2, and the acetylated N-terminus contributes nothing, so the net charge at neutral pH is close to zero while the chain remains polar throughout. The peptide is highly water soluble. There are no aromatic residues, so detection is at 210 to 220 nm, and there is no cysteine, tryptophan or methionine; the oxidation-prone Met6 of the parent protein lies outside the fragment. Acetylation blocks aminopeptidases, but the free C-terminus is open to carboxypeptidases in serum-containing medium, and the C-terminal glutamine can deamidate slowly to glutamate.
In the complex between Tβ4 and monomeric actin, the N-terminal helix of the protein sits in the hydrophobic cleft at the barbed end of the monomer, the LKKTET segment runs across the face of the monomer, and the C-terminal helix caps the pointed end, so that both ends of the actin subunit are blocked from adding to a filament. The heptapeptide reproduces only the central contact.
Mechanism of action
Tβ4 is the main G-actin sequestering protein in most cell types, present at cytoplasmic concentrations in the hundreds of micromolar. It binds ATP-loaded G-actin 1:1 with a dissociation constant in the low micromolar range and holds the monomer in a form that cannot polymerise. Profilin competes for the same monomer and hands it to formins and to free barbed ends, so exchange between Tβ4 and profilin sets the size of the assembly-competent actin pool. The isolated LKKTET segment binds actin at the same site as the corresponding segment of the parent protein but with far lower affinity, and in pyrene-actin assays it slows polymerisation only at high concentrations. The fragment is therefore used as a competitor and a probe of the actin-binding site as much as a sequestering agent in its own right.
Cell migration is the second readout. Work that mapped the activities of Tβ4 to short segments found that the central actin-binding segment (residues 17 to 23) matched the full protein in promoting cell migration in scratch assays with corneal epithelial cells, while the N-terminal tetrapeptide Ac-SDKP did not. The migration response is read out as closure of the cell-free gap over hours and is accompanied by reorganisation of F-actin at the leading edge, visible with phalloidin staining. How a peptide that binds actin only weakly produces this response has not been settled; direct entry into cells and competition with endogenous Tβ4 for actin, and an extracellular receptor-mediated route, have both been proposed.
The parent protein has other reported interactions: binding to the β subunit of cell-surface ATP synthase on endothelial cells, association with integrin-linked kinase (ILK) and PINCH in the focal adhesion complex with downstream Akt phosphorylation, and an N-terminal tetrapeptide, Ac-SDKP, released by prolyl oligopeptidase. Most of these map to regions of the protein outside residues 17 to 23 or have not been tested with the heptapeptide, so they should not be assumed to carry over.
Research applications
- Pyrene-actin polymerisation kinetics, with the fragment as a low-affinity competitor of full-length Tβ4 and of WH2 domains
- G-actin binding by isothermal titration calorimetry, fluorescence anisotropy or native gel shift
- Scratch and transwell migration assays with epithelial cells, fibroblasts and endothelial cells
- F-actin imaging by phalloidin staining or live-cell actin reporters after peptide exposure
- Structure-activity comparisons with Ac-SDKP and full-length Tβ4 for mapping activity to regions of the protein
- LC-MS/MS method development, where the heptapeptide and its truncation products have been characterised as analytes
TB-500 sits in the Tissue Repair Signalling category with the other peptides used in cell migration and cytoskeleton work.
Handling in the laboratory
The lyophilised powder is reconstituted with bacteriostatic water. It dissolves almost at once; run the water down the inside of the vial and swirl. The reconstitution calculator gives the concentration that a chosen water volume produces from the vial content. For actin assays the stock is diluted into the polymerisation buffer on the day of use.
Keep the sealed vial dry, away from light and refrigerated as described in the product documentation. Once reconstituted, keep the solution refrigerated and use it within the period stated on the documentation, and aliquot to avoid repeated freeze-thaw cycles. The compound is characterised by RP-HPLC for purity and by mass spectrometry for identity. On electrospray the singly charged ion is observed near m/z 890 and the doubly charged ion near m/z 445; a peak near m/z 848 would indicate the non-acetylated sequence, which is 42 Da lighter.
Testing and supply from NuVion
NuVion has TB-500 independently tested by Janoshik Analytical. Most batches are tested, and the Certificate of Analysis for a tested batch is published on the product page, reporting purity by RP-HPLC and identity by mass spectrometry. Manufacture is GMP-audited, the peptide is supplied lyophilised in sealed vials, and orders are dispatched from within Australia. Certificates for other compounds are collected in the Certificate of Analysis library.
Related compounds
TB-500 is one of the four peptides in the KLOW blend, alongside GHK-Cu, BPC-157 and KPV, where it supplies actin sequestration. The parent protein has its own profile in the library, thymosin beta-4. Two other peptides in the same category are used in overlapping assays: BPC-157, the gastric pentadecapeptide used in migration and focal adhesion signalling work, and GHK-Cu, the copper-binding tripeptide used in fibroblast and extracellular matrix gene expression studies. Ligand traps that remove myostatin and activin from the same tissue context are covered in follistatin and ACE-031.
Frequently asked questions
What is TB-500 used for in research?
TB-500 is used in actin biochemistry as a competitor and probe of the Tβ4 binding site on G-actin, in pyrene-actin polymerisation and binding assays, and in cell migration and cytoskeleton imaging work with epithelial, fibroblast and endothelial cultures. It is also a characterised analyte in LC-MS/MS method development.
Is TB-500 the same as thymosin β4?
No. Thymosin β4 is the 43-residue protein; TB-500 as supplied here is the N-acetylated seven-residue fragment covering residues 17 to 23, which carries the LKKTET actin-binding motif. The two differ in mass by a factor of about five and in actin affinity by a large margin, and the name TB-500 is sometimes applied to the full protein, so the Certificate of Analysis mass is the check.
How is TB-500 supplied and stored?
NuVion supplies TB-500 as a lyophilised powder in a sealed vial. Store the sealed vial dry, away from light and refrigerated per the product documentation. After reconstitution with bacteriostatic water, keep the solution refrigerated and use it within the period given on the documentation.
Is TB-500 a therapeutic good in Australia?
No. TB-500 from NuVion is a laboratory chemical for in vitro research. It is not included in the Australian Register of Therapeutic Goods and has not been assessed by the TGA. It is not for human or veterinary use.
Research use only. This product is a laboratory chemical supplied for in vitro research. It is not included in the Australian Register of Therapeutic Goods and has not been assessed by the Therapeutic Goods Administration for quality, safety or efficacy. It is not for human or veterinary use, and nothing on this page is a representation about therapeutic use.
