Researcher positioning a cell-culture dish beneath a laboratory microscope

Healing · Laboratory protocol

TB-500

Thymosin beta-4 related material, studied for actin sequestration, cell migration and angiogenesis. Structurally unrelated to BPC-157 despite the two being routinely discussed together.

For laboratory research use only. Material is supplied as a research reference standard and is not approved by Health Canada. Protocols below are compiled from published literature and supplier documentation; they are reference information, not medical advice. Anyone with a health condition or on medication should speak to a physician first.

Before you begin

• Lot-matched certificate and laboratory notebook
• Calibrated pipettes, low-binding tubes and suitable PPE
• Validated solvent, assay reagents and model-specific SOP
• Appropriate positive, negative and vehicle controls

Workflow

1Review the lot-matched COA and record identity, purity, batch number and received condition.
2Let the sealed vial equilibrate to room temperature before opening to limit condensation.
3Calculate the target stock concentration with C = mass / volume. Use only a solvent validated for the planned assay.
4Add solvent slowly down the vial wall, mix gently and avoid vortexing unless the validated method permits it.
5Prepare a log-spaced pilot concentration series; determine the usable range empirically instead of assuming a biological dose.
6Run the defined controls and technical replicates alongside every plate or experiment.
7Record preparation time, temperature, equipment, deviations and raw-data file locations.

Assay design

Endothelial tube-formation or migration endpoints, with the actin-binding mechanism probed separately rather than inferred from a closure rate.

Required controls

Vehicle-only negative control
Untreated time-zero control
Validated migration control
Parallel cytotoxicity or viability assay

Reported protocol

Loading phase: 2.0 mg to 5.0 mg twice weekly for 4 to 6 weeks
Maintenance phase: 2.0 mg every 2 weeks
Route: Subcutaneous or intramuscular

Diluent

Sterile bacteriostatic water (0.9% benzyl alcohol) for multi-withdrawal stock, or sterile water for injection where a preservative-free stock is required by the assay.

Reconstitution concentrations

Solvent volume sets the stock concentration for this 10 mg vial. Lower volume gives a more concentrated stock and smaller working aliquots; higher volume gives a more dilute stock that is easier to measure accurately.

Stock concentration by solvent volume for a 10 mg vial
Solvent added	Stock concentration	Per 0.1 mL	Per unit (U-100)
1 mL	10 mg/mL	1 mg	100 mcg
2 mL	5 mg/mL	500 mcg	50 mcg
3 mL	3.33 mg/mL	333.33 mcg	33.33 mcg
5 mL	2 mg/mL	200 mcg	20 mcg

Working range

In-vitro migration and tube-formation work typically runs the nanomolar to low-micromolar band.

Endothelial tube-formation or migration endpoints, with the actin-binding mechanism probed separately rather than inferred from a closure rate.

Stability

Lyophilized: Stable sealed and dry at the certificate temperature.
In solution: Establish empirically; aliquot to avoid repeated freeze-thaw.

Handling notes

Confirm on the lot certificate whether the material is the full-length sequence or a fragment. The two are not interchangeable and the literature uses both names loosely.
Use low-binding tubes for dilute stocks.
If comparing against BPC-157, run both on the same plate; they are separate molecules with separate literatures.

Documentation checklist

Record lot and COA, mass balance, solvent and concentration, preparation timestamp, storage history, plate map, replicate plan, instrument settings, exclusions and deviations. Preserve raw data and analysis code with the experiment record.