Recombinant Human TAFA-2

Recombinant Human TAFA-2 offers several compelling advantages for research applications, particularly in neurobiology, stem cell biology, and tissue regeneration studies.

Biological Significance and Research Applications

TAFA-2 functions as a neurotrophic factor essential for neuronal survival and neurobiological functions. The protein is predominantly expressed in the central nervous system, with mRNA expression levels 50- to 1000-fold higher in the CNS compared to peripheral tissues. This CNS-specific expression pattern makes it particularly valuable for investigating neurological processes.

The protein exhibits multiple biological roles that expand its research utility. TAFA-2 acts as a brain-specific chemokine or neurokine, potentially regulating immune and nervous system interactions. Beyond neuronal applications, TAFA-2 demonstrates significant activity in stem cell biology, where it stimulates human mesenchymal stem cell (hMSC) migration through activation of the Rac1-p38 signaling pathway. This property is particularly relevant for bone regeneration research, as TAFA-2 expression increases during the inflammatory phase of fracture healing.

Technical Advantages

Using recombinant TAFA-2 provides several practical benefits for experimental work:

High purity and consistency: Recombinant protein technology enables production of proteins with standardized quality and reproducibility, essential for obtaining reliable experimental results across multiple studies.

Defined molecular characteristics: Recombinant Human TAFA-2 is a non-glycosylated polypeptide containing 101 amino acids with a molecular weight of approximately 11.2 kDa. The protein demonstrates high sequence conservation between species, with 97% amino acid identity between human and mouse variants, facilitating translational research.

Availability in multiple formats: The protein is available in various preparations, including lyophilized forms and carrier-free or BSA-supplemented formulations, allowing researchers to select the most appropriate format for their specific experimental protocols.

Research Model Relevance

Studies using TAFA-2 knockout mice have demonstrated its importance in spatial learning, memory formation, and anxiety-related behaviors, providing a strong biological rationale for investigating this protein in neuroscience research. The conservation of TAFA-2 function across mammalian species supports its use as a research tool for understanding fundamental neurobiological mechanisms applicable to human physiology.

Recombinant Human TAFA-2 can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity, properly quantified, and compatible with your assay system. This is a common practice in quantitative ELISA, where recombinant proteins serve as standards to generate a calibration curve for determining the concentration of the target analyte in samples.

Key considerations for using recombinant TAFA-2 as an ELISA standard:

• Purity and Characterization: The recombinant TAFA-2 should be highly pure (typically >98% by SDS-PAGE and HPLC) and well-characterized, with low endotoxin levels, to ensure accurate quantification and avoid interference in the assay.
• Quantification: The protein concentration must be accurately determined, usually by absorbance at 280 nm or another validated method, to prepare precise standard dilutions.
• Compatibility: The recombinant TAFA-2 should be recognized by the capture and detection antibodies used in your ELISA. Most commercial TAFA2 ELISA kits are designed to detect both natural and recombinant forms of human TAFA-2, but this should be confirmed for your specific assay.
• Standard Curve Preparation: Prepare a standard curve by serially diluting the recombinant TAFA-2 in the same matrix as your samples (e.g., serum, plasma, or buffer) to account for potential matrix effects.
• Validation: It is essential to validate the linearity, accuracy, and precision of your standard curve within the assay’s dynamic range. Ensure that the recombinant standard behaves similarly to endogenous TAFA-2 in your sample matrix.

Additional best practices:

• Use carrier-free or BSA-containing formulations as appropriate for your application; carrier-free is preferred for ELISA standards to avoid interference.
• Store the recombinant protein as recommended (typically at −20°C or lower) to maintain stability.
• If using a home-prepared standard, ensure rigorous quality control, as impurities or inaccurate quantification can compromise assay accuracy.

Summary:
You can use recombinant human TAFA-2 as a standard in ELISA assays if it is pure, accurately quantified, and validated for your assay system. This approach is standard in quantitative ELISA protocols for cytokines and secreted proteins. Always confirm compatibility with your specific antibodies and validate the standard curve for reliable quantification.

Recombinant Human TAFA-2 (also known as FAM19A2) has been validated for several key applications in published research, primarily centered on its biological activity in cell migration, neurotrophic effects, and signaling pathway modulation. The main applications supported by peer-reviewed studies include:

1. Enhancement of Skeletal (Mesenchymal) Stem Cell Migration

   • TAFA-2 has been shown to stimulate the migration and motility of human mesenchymal stem cells (hMSCs) in a dose-dependent manner, as demonstrated in trans-well migration assays and Oris assays.
   • It induces morphological changes such as lamellipodia formation, which are critical for cell movement.
   • The mechanism involves activation of the Rac1-p38 signaling pathway.
   • This application is relevant for regenerative medicine, particularly in bone tissue repair and fracture healing.
   • Reference: PMC7379704

2. Neurotrophic and Neuronal Survival Effects

   • TAFA-2 functions as a neurotrophic factor, supporting neuronal survival and neurobiological functions.
   • It has been validated for its ability to enhance neurite outgrowth in rat embryonic cortical neurons.
   • Reference: PMC6185136, Cell Sciences product PDF

3. Modulation of Metabolic Activities

   • Studies indicate that TAFA-2 participates in the regulation of food intake and metabolic activities, suggesting a role in metabolic research.
   • Reference: PubMed 31220516

4. Immune Modulation in the Central Nervous System

   • TAFA-2 is postulated to act as a brain-specific chemokine or neurokine, modulating immune responses in the CNS.
   • Reference: Nature, Bio-Techne product information

5. In Vivo Fracture Healing Studies

   • In animal models, TAFA-2 overexpression at fracture sites increases recruitment of MSCs to injured tissue, supporting its use in studies of bone regeneration and repair.
   • Reference: PMC7379704

These applications highlight TAFA-2’s utility in stem cell biology, neuroscience, regenerative medicine, and metabolic research. Most recombinant forms are validated for use in cell culture, migration assays, neurite outgrowth assays, and in vivo studies, but not for clinical diagnostic or therapeutic use.

To properly reconstitute and prepare Recombinant Human TAFA-2 protein for cell culture experiments, follow these evidence-based steps:

Reconstitution Protocol

1. Equilibrate and Centrifuge
   Allow the lyophilized vial and your reconstitution buffer to reach room temperature. Briefly centrifuge the vial (or tap it down if a centrifuge is unavailable) to ensure all the lyophilized powder is at the bottom.

2. Reconstitution Buffer
   Reconstitute the protein in sterile distilled water or an aqueous buffer containing 0.1% BSA (bovine serum albumin). This helps stabilize the protein and prevent adsorption to surfaces.

3. Concentration
   Add the buffer to achieve a concentration of 0.1–1.0 mg/mL, depending on your experimental needs. Refer to the product datasheet or Certificate of Analysis for specific recommendations.

4. Reconstitution Method
   Gently swirl or invert the vial to dissolve the protein. Avoid vigorous shaking to prevent foaming and protein denaturation. Allow the solution to reconstitute for 15–30 minutes at room temperature with gentle agitation. If flakes or particulates remain, mix for a couple of hours at room temperature, then at 4°C overnight.

5. Aliquoting
   Once fully dissolved, aliquot the reconstituted protein into working volumes (preferably >20 µL) to minimize freeze-thaw cycles.

Storage and Handling

• Short-term (1 week): Store the reconstituted protein at 2–8°C.
• Long-term: Store aliquots at ≤ –20°C (preferably in a manual defrost freezer). Avoid repeated freeze-thaw cycles.
• Lyophilized protein: Store desiccated at –20°C for long-term stability.

Preparation for Cell Culture

• Dilution: Dilute the reconstituted protein in appropriate cell culture medium or buffer. If your experiment requires serum-free conditions, avoid carrier proteins like BSA and consider using alternatives such as trehalose as a stabilizer.
• Working Dilution: Each investigator should determine the optimal working dilution for their specific cell type and application.

Additional Notes

• Endotoxin: Ensure the protein is low in endotoxin (<1 EU/µg) for sensitive cell culture applications.
• Stability: The reconstituted protein is stable for up to one week at 2–8°C and for several months when stored in aliquots at –20°C or lower.

Always refer to the specific product datasheet or Certificate of Analysis for any unique requirements or recommendations.