Thymosin Alpha-1 is a 28-residue, N-terminally acetylated peptide corresponding to the N-terminal fragment of prothymosin alpha. It was first isolated from thymosin fraction 5, a thymic tissue extract, and is now made by solid-phase synthesis. In the laboratory it is used as a tool compound in Toll-like receptor signalling assays, in dendritic cell and thymocyte culture work, and as a reference peptide in chromatography and mass spectrometry method development. NuVion supplies Thymosin Alpha-1 as a laboratory chemical for in vitro research use only.
Key facts
| Type | Synthetic peptide, N-terminally acetylated fragment of prothymosin alpha |
| Amino acid count | 28 |
| Sequence | Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH (Ac-SDAAVDTSSEITTKDLKEKKEVVEEAEN) |
| Molecular weight | Approximately 3,108 g/mol |
| CAS number | 62304-98-7 |
| Synonyms | Thymalfasin, T-alpha-1, thymosin alpha 1, prothymosin alpha(1-28) |
| Supplied form | Lyophilised powder in a sealed vial |
Thymosin Alpha-1
$109 AUD
View productResearch use only. Not for human or veterinary use.
Structure and chemistry
Prothymosin alpha is a small, highly acidic nuclear protein. Thymosin Alpha-1 is its first 28 residues, carrying an acetyl group on the alpha-amino group of the N-terminal serine. That acetylation is part of the definition of the molecule, so a synthetic preparation with a free N-terminus is a different compound and behaves differently on ion-exchange chromatography and in mass spectrometry, where the acetyl group adds 42 Da to the expected mass.
The sequence is strongly acidic. It carries three aspartate and six glutamate side chains against four lysines, and because the N-terminus is blocked it contributes no positive charge, so the peptide is net negative at neutral pH. There is no arginine and no histidine. There is also no cysteine, so the peptide forms no disulfide bonds and no intramolecular bridge, and no methionine, so oxidation is not a normal degradation route. The sequence contains no tryptophan, tyrosine or phenylalanine, which means the peptide has essentially no absorbance at 280 nm; concentration is determined at 214 nm from the peptide bond, gravimetrically, or from the value on the certificate.
Circular dichroism describes the peptide as largely disordered in aqueous buffer. Helical content appears in membrane-mimicking solvents and in the presence of lipid, which is consistent with a peptide that is flexible in solution and takes up structure on contact with a surface. The acidic side chains make it readily soluble in water and in neutral buffers, and it elutes early on a reversed-phase gradient relative to hydrophobic peptides of similar length. Points to watch on stability are deamidation at the C-terminal asparagine and, over long periods in solution, hydrolysis at the Asp-Ala bonds.
Mechanism of action
No single high-affinity receptor has been defined for Thymosin Alpha-1. The activity described in cell culture is instead attributed to engagement of Toll-like receptors, chiefly TLR2 and TLR9, on antigen-presenting cells. Both receptors signal through the adaptor MyD88, which recruits IRAK4 and IRAK1 and then TRAF6. That cascade branches: the IκB kinase complex is activated, IκBα is phosphorylated and degraded, and NF-κB dimers containing p65 move to the nucleus, while in parallel the MAP kinases p38 and JNK are phosphorylated. In monocyte-derived dendritic cell cultures exposed to the peptide, p65 nuclear translocation and p38 phosphorylation are both detectable, and transcription of the cytokine genes downstream of those factors changes accordingly.
Two downstream readouts are used most often in dendritic cell work. The first is expression of IL-12 subunits and of type I interferon genes, measured by qPCR or by ELISA on culture supernatant, which is the transcriptional signature associated with a Th1-type polarising phenotype. The second is induction of indoleamine 2,3-dioxygenase (IDO1), the enzyme that converts tryptophan to kynurenine; IDO1 induction in these cultures is interferon-dependent and is read out either as protein by Western blot or as enzyme activity by measuring kynurenine in the medium. Because TLR2 and TLR9 have well-characterised reference ligands (bacterial lipopeptides and CpG oligodeoxynucleotides), the peptide is often run alongside them so that the receptor dependence of a response can be tested directly, including in TLR-transfected reporter lines and in cells where the receptor or MyD88 has been knocked down.
A separate and older line of cell work concerns thymocyte and T-cell maturation. In thymocyte and T-cell line culture, exposure to the peptide changes the expression of surface markers associated with lineage commitment, including CD3, CD4 and CD8, and of terminal deoxynucleotidyl transferase, which is a marker of immature thymocytes. These are flow cytometry and enzyme-assay readouts at the level of the cultured cell, and they are the basis for describing the peptide as a modulator of T-cell differentiation. The molecular link between TLR engagement on antigen-presenting cells and the thymocyte observations has not been resolved.
Research applications
Thymosin Alpha-1 sits in NuVion’s Immune Signalling category. Typical study contexts include:
- NF-κB and interferon-stimulated response element reporter assays in HEK293 lines transfected with TLR2 or TLR9, with reference ligands as comparators
- Monocyte-derived dendritic cell culture: surface marker phenotyping by flow cytometry, cytokine gene expression by qPCR, cytokine release by ELISA
- IDO1 induction and kynurenine accumulation assays, with MyD88 or TLR knockdown to establish pathway dependence
- Western blotting of p65 nuclear fractions, IκBα degradation and p38 or JNK phosphorylation in antigen-presenting cell lines
- Thymocyte and T-cell line culture, tracking differentiation markers and terminal deoxynucleotidyl transferase activity
- Analytical work: RP-HPLC method development for a long, strongly acidic peptide, and LC-MS confirmation of the acetylated N-terminus and of deamidation products
Handling in the laboratory
The peptide is supplied as a lyophilised powder in a sealed vial. Reconstitute with bacteriostatic water, directing the stream down the vial wall so the powder wets without foaming; the volume for a given working concentration can be worked out with the reconstitution calculator. Being an acidic peptide it dissolves quickly in water and in neutral buffers. Keep the sealed vial dry and away from light, refrigerated as stated in the product documentation. Refrigerate the reconstituted solution and use it within the period given on the documentation, and divide it into single-use aliquots if the work spans several sessions, so the stock avoids repeated freeze-thaw. Since deamidation at the C-terminal asparagine is the main slow change in solution, keep the pH neutral where the experiment allows. The compound is characterised by RP-HPLC for purity and by mass spectrometry for identity.
Testing and supply from NuVion
NuVion’s Thymosin Alpha-1 comes from GMP-audited manufacture and is independently tested by Janoshik Analytical. Most batches are tested, and the Certificate of Analysis for a tested batch, giving RP-HPLC purity and mass spectrometry identity, is published on the product page and in the certificates of analysis library. It is supplied lyophilised in sealed vials and dispatched from within Australia.
Related compounds
Two other thymus-derived compounds in the same category are Thymulin, a nonapeptide whose activity in assays depends on bound zinc, and Thymogen, the Glu-Trp dipeptide. Both are shorter sequences studied in the same kind of cell-based signalling work.
Frequently asked questions
What is Thymosin Alpha-1 used for in research?
It is used as a tool compound in Toll-like receptor signalling assays, particularly TLR2 and TLR9 reporter systems and dendritic cell cultures, where readouts include NF-κB translocation, cytokine gene expression and IDO1 induction. It is also used in thymocyte and T-cell line differentiation work, and as a reference peptide for analytical method development.
Why does the acetyl group matter?
The native molecule is acetylated on the N-terminal serine, so an unacetylated 28-mer of the same sequence is a different compound. The acetyl group removes the N-terminal positive charge and adds 42 Da to the mass, which is how mass spectrometry confirms that the correct form is present.
Is Thymosin Alpha-1 a therapeutic good in Australia?
No. NuVion supplies it as a laboratory chemical for in vitro research, and it is not for human or veterinary use. The regulatory position is set out in the research use only notice at the end of this article.
How should Thymosin Alpha-1 be stored?
Keep the sealed vial of lyophilised powder dry, away from light and refrigerated as stated in the product documentation. After reconstitution with bacteriostatic water, refrigerate the solution and use it within the period on the documentation. Aliquot the solution if repeated sampling is planned so the stock is not put through freeze-thaw cycles.
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.
