Transforming growth factor-beta 3 (TGF-β3), also known as TGFB3, is a member of the TGF beta family of growth factors along with TGF-β1 and -2. The members of this family can be expressed by most cell types and are proposed to act as cellular switches that regulate immune function, proliferation and epithelial-mesenchymal transition (1). These cytokines are secreted in precursor form consisting of a bioactive C-terminal domain attached to an N-terminal domain known as latency associated protein (LAP). Cleavage of LAP results in the mature protein, which functions as a disulfide-linked homodimer. TGF-β3 and these homodimers of LAP remain non-covalently associated after secretion, forming the latent TGF-β3 complex (2). Activation of this latent complex is accomplished by actions from plasmin, MMPs, thrombospondin 1 and some integrins (3). The receptor for TGF-β3 is TGF-β RII which phosphorylates, and activates another receptor, either ALK-5 or ALK-1. This second complex activates Smad proteins that regulate transcription. TGF-β3 is involved in oxygen-dependent differentiation processes during placental development and pregnancy disorders (4) and it also plays an important role in wound repair and scarring (5). TGF-β3 is believed to regulate molecules involved in cellular adhesion and extracellular matrix (ECM) formation during the process of palate development, without it, mammals develop a deformity known as a cleft palate (6). Similarly, TGF-β3 also plays an essential role in controlling the development of lungs in mammals, also by regulating cell adhesion and ECM formation in this tissue (6). Defects in the TGF-β3 gene are a cause of familial arrhythmogenic right ventricular dysplasia 1 (ARVD1) (7). ARVD1 is an autosomal dominant disease characterized by partial degeneration of the myocardium of the right ventricle, electrical instability and sudden death.
Protein Details
Purity
>97% by SDS-PAGE and analyzed by silver stain.
Endotoxin Level
<0.01 EU/µg as determined by the LAL method
Biological Activity
The biological activity of Human TGF-β3 was determined by its ability to inhibit the IL-4-dependent proliferation of mouse HT-2 cells. The expected ED<sub>50</sub> for this effect is typically 0.03 - 0.08 ng/ml.
The predicted molecular weight of Recombinant Human TGF-β3 is Mr 12.7 kDa. However, the actual molecular weight as observed by migration on SDS-PAGE is 12 kDa (reducing conditions) and 24 kDa (non-reducing conditions).
Predicted Molecular Mass
12.7
Formulation
This recombinant protein was lyophilized from a 0.2 μm filtered solution in 40% acetonitrile (CH3CN) and 0.1% trifluoroacetic acid (TFA).
Storage and Stability
This lyophilized protein is stable for six to twelve months when stored desiccated at -20°C to -70°C. After aseptic reconstitution, this protein may be stored at 2°C to 8°C for one month or at -20°C to -70°C in a manual defrost freezer. Avoid Repeated Freeze Thaw Cycles. See Product Insert for exact lot specific storage instructions.
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Recombinant Human TGF-β3 is widely used in research because it is a potent, well-characterized growth factor that regulates key cellular processes such as differentiation, proliferation, tissue regeneration, and immune modulation, making it essential for studies in stem cell biology, tissue engineering, wound healing, and developmental biology.
Key scientific reasons to use recombinant human TGF-β3 in your research applications include:
Stem Cell Differentiation: TGF-β3 is critical for directing the differentiation of mesenchymal stem cells (MSCs) into chondrocytes (cartilage cells) and osteoblasts (bone cells), making it indispensable for studies on chondrogenesis and osteogenesis. It enhances the production of type II collagen and aggrecan, key markers of cartilage, and is used in protocols for generating cartilage tissue from stem cells.
Tissue Engineering and Regeneration: TGF-β3 promotes tissue regeneration and matrix formation, supporting applications in cartilage repair, bone healing, and wound healing models. It has been shown to recruit endogenous stem cells to sites of injury and enhance their regenerative capacity.
Developmental Biology: TGF-β3 is essential for embryonic development, including palate and lung formation, and is used to study mechanisms of organogenesis and tissue patterning.
Immunomodulation: TGF-β3 has unique anti-inflammatory properties, such as inhibiting T cell and B cell proliferation and modulating regulatory T cell function, which distinguishes it from other TGF-β isoforms and is valuable for immunology research.
Wound Healing: It accelerates wound closure and improves tissue remodeling, making it a model factor for studying mechanisms of scarless healing and fibrosis.
Signal Transduction Studies: TGF-β3 is a key ligand for investigating the TGF-β receptor/Smad signaling pathway, which is central to many cellular responses and disease models.
Technical advantages of recombinant human TGF-β3:
High purity and consistent biological activity, ensuring reproducibility in cell culture and functional assays.
Animal-free and endotoxin-free preparations are available, reducing variability and risk of contamination in sensitive applications.
Suitable for a range of assays, including ELISA, Western blotting, inhibition assays, and functional cell-based assays.
In summary, recombinant human TGF-β3 is a versatile and reliable tool for research in cell biology, regenerative medicine, immunology, and developmental biology due to its well-defined roles in cell differentiation, tissue regeneration, and immune regulation.
Yes, recombinant human TGF-β3 can be used as a standard or calibrator for quantification in ELISA assays, provided it is suitable for your specific ELISA format and detection system.
Key Points:
Purpose of Recombinant Standards: Recombinant human TGF-β3 is commonly used to generate a standard curve in ELISA assays. This curve allows you to interpolate the concentration of TGF-β3 in your unknown samples based on the signal intensity.
ELISA Compatibility: Most commercial ELISA kits and development systems (such as DuoSet ELISA kits) include a recombinant TGF-β3 standard for this purpose. If you are using a kit, check the manufacturer’s instructions to confirm compatibility.
Formulation Considerations: Some recombinant TGF-β3 proteins are supplied with carrier proteins (e.g., BSA), which may affect assay performance if not accounted for. For best results, use a carrier-free version if possible, or ensure that your assay buffer and diluents are compatible.
Bioactivity vs. Quantification: While recombinant TGF-β3 is suitable for ELISA calibration, it is generally not recommended for bioassays unless specifically validated for such use.
Recommendations:
Use a recombinant human TGF-β3 protein that is purified and quantified (e.g., lyophilized with known concentration).
Follow the reconstitution and dilution instructions provided by the supplier.
Prepare a serial dilution of the standard to cover the expected range of your samples.
Ensure that the standard is compatible with your ELISA detection antibodies and assay conditions.
In summary, recombinant human TGF-β3 is appropriate for use as a standard in ELISA assays for quantification, as long as it is properly validated for your specific application.
Recombinant Human TGF-β3 has been validated for a broad range of applications in published research, primarily in studies involving cell signaling, differentiation, and functional assays. The most commonly validated applications include:
Functional Assays: Used to assess biological activity, such as inhibition of IL-4-induced proliferation in HT-2 cells, and to study TGF-β3’s effects on cell signaling pathways, proliferation, and differentiation.
Cell Culture and Differentiation Studies: Extensively used to direct the differentiation of mesenchymal stem cells, particularly in chondrogenesis (cartilage formation), osteogenesis (bone formation), and studies of tissue engineering and organ development.
Bioassays: Validated in various bioassays to measure its activity in modulating immune responses, stem cell pluripotency, and lineage commitment.
ELISA (Enzyme-Linked Immunosorbent Assay): Used as a standard or control protein in ELISA-based detection and quantification of TGF-β3.
Western Blot: Applied as a positive control or for detection of TGF-β3 in protein extracts.
Blocking Assays: Used to block or neutralize TGF-β3 activity in functional studies, helping to dissect its specific biological roles.
Immunohistochemistry: Utilized for localization and detection of TGF-β3 in tissue sections.
Key research applications and contexts include:
Stem Cell Biology: Induction of chondrogenic and osteogenic differentiation in mesenchymal stem cells, and maintenance of pluripotency in human pluripotent stem cells.
Developmental Biology: Investigation of roles in embryogenesis, palatogenesis, and organogenesis.
Wound Healing and Tissue Engineering: Studied for its ability to promote wound healing and tissue regeneration.
Immunology: Modulation of immune cell proliferation and differentiation, including effects on T cell responses.
Cancer and Disease Models: Used to study TGF-β signaling in cancer, fibrosis, and other pathologies.
Neuroscience: Applied in protocols for differentiation of dopaminergic neurons from human pluripotent stem cells.
Summary Table of Validated Applications
Application Type
Description/Context
Functional Assay
Bioactivity, cell signaling, inhibition of proliferation
Cell Culture
Differentiation of stem cells, tissue engineering
Bioassay
Activity quantification, immune modulation
ELISA
Standard/control for quantification assays
Western Blot
Detection/validation of TGF-β3 protein
Blocking Assay
Functional blocking/neutralization studies
Immunohistochemistry
Localization in tissue sections
These applications are supported by both product validation data and peer-reviewed research, demonstrating the versatility of recombinant human TGF-β3 in basic and translational biomedical research.
To reconstitute and prepare Recombinant Human TGF-β3 for cell culture experiments, dissolve the lyophilized protein in sterile 4 mM HCl containing a carrier protein such as 0.1–1% human or bovine serum albumin (HSA or BSA) to a concentration typically between 20–100 μg/mL. This approach minimizes protein loss and preserves activity.
Step-by-step protocol:
Centrifuge the vial briefly to collect all lyophilized material at the bottom before opening.
Add sterile 4 mM HCl to the vial. The recommended reconstitution concentration is:
20–50 μg/mL for most applications.
For higher stability, you may prepare a stock solution at 50–100 μg/mL.
Include carrier protein: Add 0.1–1% endotoxin-free HSA or BSA to the diluent to prevent adsorption and enhance stability.
Gently mix by swirling or tapping. Avoid vigorous pipetting or vortexing, which can denature the protein.
Aliquot the reconstituted solution to avoid repeated freeze-thaw cycles.
Storage:
Store at 4°C for up to 1 week.
For longer-term storage, freeze aliquots at –20°C to –70°C.
Avoid repeated freeze-thaw cycles to maintain activity.
Preparation for cell culture:
Before use, dilute the stock solution further in cell culture medium to the desired working concentration, ensuring the final HCl and carrier protein concentrations are compatible with your cells.
If using a carrier-free preparation, ensure the absence of BSA/HSA is suitable for your assay, as some applications may require strictly defined conditions.
Key notes:
Always consult the specific Certificate of Analysis (CoA) or product datasheet for your batch, as optimal reconstitution conditions may vary slightly between manufacturers or lots.
If the protein is supplied without a carrier, adding a carrier protein during reconstitution is strongly recommended for cell culture applications to prevent loss due to adsorption.
Summary Table:
Step
Recommended Condition
Reconstitution
4 mM HCl + 0.1–1% HSA/BSA
Concentration
20–100 μg/mL (stock); dilute as needed for assay
Mixing
Gentle swirling/tapping
Storage (short-term)
4°C, up to 1 week
Storage (long-term)
–20°C to –70°C, aliquoted
Freeze-thaw cycles
Avoid repeated cycles
This protocol ensures maximal stability and biological activity of recombinant TGF-β3 for cell culture experiments.
References & Citations
1. Sporn, MB. et al. (2006) Cytokine Growth factor Rev. 17:3 2. Derynck, R. et al. (1988) EMBO J. 7:3737 3. Oklu, R. et al. (2000) Biochem. J. 352:601 4. Wenger, RH. et al. (2003) Placenta 24:941 5. Bandyopadhyay, B. et al. (2006) J. Cell. Biol. 172:1093 6. Kaartinen, V. et al. (1995) Nat. Genet. 11:415 7. Nattel, S. et al. (2005) Cardiovasc. Res. 65:302
Products are for research use only. Not for use in diagnostic or therapeutic procedures.
