Recombinant Human TGF-β1 (Insect Cells)

Recombinant Human TGF-β1 produced in insect cells is widely used in research because it provides a highly pure, biologically active, and consistent source of this multifunctional cytokine, which is essential for studying cell growth, differentiation, immune regulation, and tissue remodeling.

Key reasons to use this reagent in your research applications:

• Biological Relevance: TGF-β1 is a pivotal cytokine involved in embryonic development, tissue repair, immune modulation, fibrosis, and cancer biology. Using recombinant TGF-β1 allows you to precisely control experimental conditions and dissect its roles in these processes.

• Consistency and Purity: Recombinant production in insect cells yields a product with high purity (often >95–97% by SDS-PAGE) and low endotoxin levels (<0.1 EU/µg), minimizing experimental variability and unwanted immune activation. Each lot is functionally validated for biological activity.

• Post-Translational Modifications: Insect cell expression systems can provide more mammalian-like post-translational modifications (such as glycosylation and correct disulfide bond formation) compared to bacterial systems, which is important for the proper folding and activity of TGF-β1. This increases the likelihood that the recombinant protein will mimic native human TGF-β1 in functional assays.

• Versatile Applications: Recombinant human TGF-β1 is used in:

  • Differentiation and maintenance of embryonic stem cells and induced pluripotent stem cells.
  • Induction of regulatory T cells (Tregs) and Th17 cells in immunology studies.
  • Wound healing, fibrosis, and tissue regeneration models.
  • Screening for inhibitors or antibodies targeting TGF-β signaling.
  • Positive controls in TGF-β detection and quantification assays.

• Batch-to-Batch Reproducibility: Recombinant production ensures a consistent supply, overcoming the variability and scarcity of native TGF-β1 isolated from biological sources.

• Research-Grade Validation: Products are typically validated in cellular assays and quality-controlled for activity, purity, and absence of contaminants.

Summary Table: Advantages of Recombinant Human TGF-β1 (Insect Cells)

In summary, using recombinant human TGF-β1 from insect cells provides a reliable, biologically relevant, and experimentally robust tool for investigating the diverse roles of this cytokine in health and disease.

Yes, you can use Recombinant Human TGF-β1 (expressed in insect cells) as a standard for quantification or calibration in ELISA assays, provided that the recombinant protein is biologically and immunologically equivalent to the native human TGF-β1 and is compatible with the antibodies used in your ELISA kit.

Key Considerations:

1. Immunological Compatibility:
   The recombinant TGF-β1 must be recognized by the capture and detection antibodies in your ELISA kit. Most commercial ELISA kits are validated for use with recombinant human TGF-β1, but it is important to confirm that the epitopes recognized by the kit’s antibodies are present in the insect cell-expressed protein. If the kit is designed for human TGF-β1, and the recombinant protein is full-length and properly folded, it should work.

2. Form of TGF-β1:

   • Latent vs. Active:
     TGF-β1 is secreted as a latent complex (bound to LAP and LTBP). Most ELISA kits measure total TGF-β1 (after acid activation) or active TGF-β1. If your kit measures total TGF-β1, you must activate your standard (and samples) by acid treatment to release active TGF-β1 from the latent complex. If your kit measures only active TGF-β1, ensure your recombinant standard is in the active form.
   • Glycosylation:
     Insect cell-expressed proteins may have different glycosylation patterns compared to mammalian (e.g., CHO) or native human TGF-β1. However, TGF-β1 is not glycosylated, so this should not be an issue.

3. Validation:

   • It is best practice to validate the recombinant standard by running a dilution series and confirming that it produces a standard curve with linearity and sensitivity comparable to the kit’s provided standard.
   • If possible, compare the recombinant standard with a known reference (e.g., kit-provided standard or native TGF-β1) to ensure accurate quantification.

4. Kit Instructions:
   Always refer to the manufacturer’s instructions for your ELISA kit. Some kits may specify the use of a particular recombinant standard or provide guidelines for alternative standards.

Summary:

• Yes, recombinant human TGF-β1 (insect cells) can be used as a standard in ELISA assays for quantification or calibration.
• Ensure the protein is in the correct form (active or latent, depending on your assay).
• Validate the standard with your kit to confirm compatibility and accurate quantification.
• Follow kit instructions for activation and dilution procedures.

If you have the specific product code or datasheet for your recombinant TGF-β1, it would be helpful to review the immunoreactivity and recommended applications.

Recombinant Human TGF-β1 produced in insect cells has been validated for several key applications in published research, primarily in cellular assays, bioactivity studies, and disease modeling.

Validated Applications in Published Research:

• Cellular Bioassays: Used to assess TGF-β1 activity in various cell types, including immune cells, fibroblasts, epithelial cells, and cancer cell lines. These assays often measure downstream signaling events such as SMAD phosphorylation, gene expression changes (e.g., KLF6, ZEB2, SNAIL1), and functional cellular responses like proliferation, differentiation, and apoptosis.

• Fibrosis and Tissue Remodeling Models: Applied in vitro to induce fibrotic responses, study extracellular matrix production, and model wound healing processes. TGF-β1 is a central mediator in fibrosis research, driving myofibroblast differentiation and collagen synthesis.

• Immunomodulation Studies: Used to investigate TGF-β1’s role in immune cell regulation, including suppression of cytokine receptor expression (e.g., IL-2 receptor), modulation of B cell function, and regulation of nitric oxide synthase (iNOS) in macrophages and other immune cells.

• Epithelial-to-Mesenchymal Transition (EMT): Validated for inducing EMT in cancer and normal epithelial cells, a process relevant to cancer metastasis and tissue regeneration. This is typically monitored by changes in cell morphology and expression of EMT markers.

• Stem Cell Culture and Differentiation: Utilized to support the culture and differentiation of pluripotent stem cells, including induced pluripotent stem cells (iPSCs), by regulating cell fate decisions and maintaining pluripotency.

• Screening of TGF-β Inhibitors and Antibodies: Used as a positive control in assays designed to test the efficacy of TGF-β pathway inhibitors, neutralizing antibodies, or engineered TGF-β proteins.

• Disease Modeling: Employed in models of cancer, fibrosis, and immune-related diseases to study TGF-β1’s role in pathogenesis and therapeutic intervention.

Supporting Details:

• The protein’s biological activity is routinely validated by its ability to induce canonical TGF-β signaling (e.g., SMAD2/3 phosphorylation) and functional cellular responses in bioassays.
• Published studies have used recombinant TGF-β1 from insect cells in both human and animal cell models, confirming cross-species activity.
• Applications span basic research (e.g., signaling pathway analysis) and translational studies (e.g., drug screening, tissue engineering).

Additional Notes:

• While most published research focuses on recombinant TGF-β1 produced in mammalian systems, insect cell-derived TGF-β1 is validated for similar applications due to its proper folding and biological activity.
• Specific protocols may vary depending on the cell type and experimental design, but typical concentrations range from picograms to nanograms per milliliter for in vitro assays.

If you require protocols or more detailed application notes for a specific cell type or experimental context, please specify.

To reconstitute and prepare Recombinant Human TGF-β1 (Insect Cells) for cell culture experiments, follow these best-practice steps:

• Centrifuge the vial briefly before opening to ensure all lyophilized protein is at the bottom.
• Reconstitute the lyophilized protein in an acidic buffer. For TGF-β1, commonly used buffers include:
  • 10 mM HCl
  • 4–40 mM acetic acid
• The recommended concentration for reconstitution is typically 0.1–1.0 mg/mL.
• Do not vortex the solution; instead, gently swirl or invert the vial to dissolve the protein, as vigorous mixing can denature TGF-β1.
• The protein may appear as a thin film; ensure complete dissolution by gentle mixing.
• After reconstitution, further dilute the protein in cell culture medium or buffer containing a carrier protein such as 0.1% BSA to prevent adsorption to plastic surfaces and loss of activity.
• Sterile technique should be used throughout to maintain protein and culture sterility.

Storage after reconstitution:

• Store aliquots at 2–8°C for up to one month or at –20°C to –70°C for longer-term storage.
• Avoid repeated freeze-thaw cycles to maintain protein stability and activity.

Summary protocol:

1. Briefly centrifuge the vial.
2. Add sterile 10 mM HCl or 4–40 mM acetic acid to achieve 0.1–1.0 mg/mL.
3. Gently mix until fully dissolved (do not vortex).
4. Dilute to working concentration in cell culture medium with 0.1% BSA.
5. Aliquot and store as needed, avoiding freeze-thaw cycles.

Note: Always consult the specific product datasheet for any manufacturer-specific recommendations, as formulations may vary. If your protein was supplied in PBS or another neutral buffer, reconstitution in water may be possible, but acidic conditions are generally preferred for TGF-β1 due to its hydrophobicity and tendency to aggregate.