Recombinant Human TAFA-2

Recombinant Human TAFA-2 is a valuable tool for research applications due to its well-characterized biological activity, high purity, and relevance in multiple physiological processes. Here are key reasons to use Recombinant Human TAFA-2 in your research:

1. High Purity and Consistency

Recombinant Human TAFA-2 is produced using recombinant protein technology, ensuring high purity (typically >95% by SDS-PAGE and HPLC) and batch-to-batch consistency. This is critical for reproducible experimental results, especially in sensitive assays such as cell culture, migration, and signaling studies.

2. Biological Activity

Recombinant TAFA-2 is fully biologically active and has been shown to:

• Enhance neurite outgrowth in rat embryonic cortical neurons, making it useful for neuroscience research.
• Promote migration and motility of human mesenchymal stem cells (hMSCs), supporting studies in regenerative medicine and bone tissue repair.
• Stimulate proliferation of hMSCs without altering their differentiation potential, which is important for stem cell biology and tissue engineering.

3. Mechanistic Insights

TAFA-2 activates specific signaling pathways, such as the Rac1-p38 pathway, which regulates cell migration and motility. Using recombinant TAFA-2 allows researchers to dissect these pathways and understand their roles in cell behavior and tissue regeneration.

4. Relevance in Disease Models

TAFA-2 has been implicated in:

• Neuronal survival and neurobiological functions, with knockout studies showing impairments in learning, memory, and anxiety-like behaviors.
• Fracture healing, where TAFA-2 expression is upregulated during the inflammatory phase, suggesting a role in tissue repair and regeneration.

5. Versatility in Applications

Recombinant TAFA-2 can be used in a variety of experimental setups, including:

• Cell-based assays (migration, proliferation, differentiation)
• In vivo studies (animal models of injury, regeneration, or neurodegeneration)
• ELISA standards and protein-protein interaction studies

6. Species Homology

Human TAFA-2 is 97% identical to mouse TAFA-2, making it suitable for cross-species studies and translational research.

7. Support for Regenerative Medicine

Given its ability to recruit stem cells to injury sites and promote tissue repair, recombinant TAFA-2 is a promising candidate for developing therapies in regenerative medicine, particularly for bone and neural tissue regeneration.

8. Ease of Use

Recombinant TAFA-2 is typically supplied in lyophilized form, which is stable and easy to reconstitute for immediate use in experiments.

In summary, Recombinant Human TAFA-2 is a reliable, biologically active protein that supports a wide range of research applications in neuroscience, stem cell biology, and regenerative medicine. Its high purity, consistent activity, and relevance in key biological processes make it an excellent choice for advancing your research.

Yes, you can use recombinant human TAFA-2 as a standard for quantification or calibration in your ELISA assays, provided it is of high purity and its concentration is accurately known. This is a common and accepted practice in quantitative ELISA protocols.

Key considerations and supporting details:

• Recombinant proteins are routinely used as ELISA standards to generate calibration curves, enabling quantification of the same protein in biological samples. The standard must be pure, stable, and its concentration precisely determined.
• Commercial ELISA kits for human TAFA-2 use recombinant human TAFA-2 as the standard to generate the assay’s calibration curve. These kits specify that the assay will recognize both natural and recombinant forms of TAFA-2, ensuring that the standard curve is valid for quantifying endogenous TAFA-2 in samples.
• Best practices for using recombinant TAFA-2 as a standard:
  • Confirm the purity (typically >95% by SDS-PAGE/HPLC) and endotoxin level (if relevant for cell-based assays) of your recombinant TAFA-2.
  • Prepare a serial dilution of the recombinant TAFA-2 in the same buffer or matrix as your samples to construct a standard curve.
  • Ensure the standard curve range matches the expected concentration range in your samples (e.g., 32.5–8000 pg/mL as used in validated TAFA-2 ELISA kits).
  • Validate that the antibodies used in your ELISA recognize both recombinant and native TAFA-2 in a parallel manner, which is standard in well-designed commercial kits.
• Carrier-free recombinant protein is preferred for ELISA standards to avoid interference from stabilizing proteins such as BSA.

Limitations and caveats:

• If you are developing a custom ELISA, you must verify that your assay’s antibodies bind the recombinant standard and the native protein equivalently.
• The accuracy of quantification depends on the quality and calibration of the recombinant standard; impure or inaccurately quantified standards can introduce error.

In summary: Using recombinant human TAFA-2 as a standard is scientifically valid and widely practiced for ELISA quantification, as long as you follow best practices for standard preparation and assay validation.

Research Applications and Validation

Recombinant Human TAFA-2 (also designated FAM19A2) has been validated for several important applications in published research, primarily centered on cellular migration, neurobiological functions, and regenerative medicine.

Mesenchymal Stem Cell Migration and Recruitment

The most extensively documented application involves enhancing human mesenchymal stem cell (hMSC) migration and recruitment to injury sites. Research has demonstrated that recombinant TAFA-2 stimulates hMSC migration in a dose-dependent manner through activation of the Rac1-p38 signaling pathway. The protein induces characteristic morphological changes including formation of lamellipodia, which are cellular protrusions essential for cell movement. This activity has been validated using multiple in vitro assays, including three-dimensional Boyden chamber assays and the Oris migration assay, with studies confirming that TAFA-2 exhibits chemotactic (directional) rather than chemokinetic (random) activity. The application is particularly relevant for bone fracture healing, where TAFA-2 gene expression is transiently upregulated during the inflammatory phase of fracture repair in animal models.

Neurite Outgrowth and Neuronal Support

Recombinant TAFA-2 has been validated for its ability to enhance neurite outgrowth in embryonic cortical neurons, with biological activity determined through its capacity to stimulate neurite extension in rat embryonic cortical neurons (E16-E18 stage). The protein functions as a neurotrophic factor essential for neuronal survival and neurobiological functions. Given that TAFA-2 mRNA expression in the human central nervous system is 50- to 1000-fold higher than in peripheral tissues, with particularly high expression in the occipital and frontal cortex and medulla, this application reflects the protein's natural biological role.

Metabolic and Physiological Regulation

Research indicates that recombinant TAFA-2 participates in the regulation of food intake and metabolic activities, suggesting broader physiological applications beyond neuronal and stem cell contexts.

Assay Development

The protein has been validated for use as an ELISA standard and in cell and tissue culture applications, with both carrier-free and BSA-supplemented formulations available for different experimental contexts.

To reconstitute and prepare Recombinant Human TAFA-2 protein for cell culture experiments, briefly centrifuge the vial to collect the lyophilized powder at the bottom, then add sterile distilled water or an aqueous buffer containing 0.1% BSA to achieve a final concentration of 0.1–1.0 mg/mL.

Step-by-step protocol:

• Equilibrate the vial and buffer to room temperature before opening.
• Centrifuge the vial briefly to ensure all powder is at the bottom.
• Add buffer: Use sterile distilled water or a buffer with 0.1% BSA. BSA acts as a carrier protein to stabilize TAFA-2 and minimize adsorption to tube walls.
• Dissolve: Gently mix by tapping or slow pipetting. Avoid vigorous shaking to prevent foaming and protein denaturation.
• Incubate: Allow the protein to dissolve for 15–30 minutes at room temperature with gentle agitation. If particulates remain, mix for a couple of hours at room temperature or overnight at 4°C.
• Aliquot: Divide the stock solution into working aliquots to avoid repeated freeze-thaw cycles.
• Storage:
  • Short-term: Store reconstituted protein at 2–8°C for up to one week.
  • Long-term: Store aliquots at ≤–20°C (preferably –80°C for maximal stability) with carrier protein.
  • Avoid repeated freeze-thaw cycles to preserve activity.

Preparation for cell culture:

• For cell culture, further dilute the stock solution in your culture medium or appropriate buffer immediately before use. If using serum-free conditions, avoid animal-derived carrier proteins and consider alternatives like trehalose.
• Confirm the final working concentration based on your experimental design; typical starting concentrations range from 10 ng/mL to 1 μg/mL, but optimization may be required for your specific cell type and assay.

Additional notes:

• TAFA-2 is a small, non-glycosylated protein (101 amino acids, ~11.2 kDa).
• Endotoxin levels are typically low (<1 EU/μg), but verify if your application is sensitive to endotoxin.
• Always consult the product-specific Certificate of Analysis for any unique requirements.

This protocol ensures maximal protein stability and biological activity for cell culture experiments.