Recombinant Human Activin RIIA

Recombinant Human Activin RIIA is used in research applications to study and manipulate signaling pathways involving the TGF-β superfamily, particularly those mediated by activins, inhibins, BMPs, and GDFs. This receptor is essential for investigating diverse biological processes such as development, neurobiology, bone metabolism, and disease mechanisms.

Key reasons to use Recombinant Human Activin RIIA in research:

• Ligand Binding and Signal Transduction Studies: Activin RIIA is a type II serine/threonine kinase receptor that binds activins and related ligands, then associates with type I receptors to initiate intracellular signaling. Using the recombinant extracellular domain allows you to dissect ligand-receptor interactions, receptor specificity, and downstream signaling events in vitro.

• Decoy Receptor and Inhibition Assays: The recombinant soluble form (often as an Fc chimera) can act as a decoy receptor, sequestering activins and related ligands, thereby inhibiting their biological activity. This is valuable for studying the functional consequences of blocking activin signaling in cell-based assays or animal models, such as in bone formation, neuroprotection, or cancer biology.

• Bioassays and ELISA Standards: Recombinant Activin RIIA is used as a standard or capture reagent in ELISA, surface plasmon resonance, and other binding assays to quantify ligand levels or receptor-ligand interactions with high specificity and reproducibility.

• Functional Studies in Development and Disease: Activin RIIA signaling is implicated in mesoderm induction, neural differentiation, bone remodeling, hematopoiesis, reproductive physiology, inflammation, and carcinogenesis. Recombinant receptor enables mechanistic studies in these contexts, including the modulation of signaling by co-receptors or antagonists.

• Therapeutic Target Validation: Recombinant Activin RIIA has been used in preclinical models to validate therapeutic strategies, such as increasing bone mass by inhibiting activin signaling with soluble receptor constructs, or exploring neuroprotective effects in models of neuronal injury.

• Protein-Protein Interaction Studies: The recombinant receptor can be used to map interactions with co-receptors (e.g., RGM-B/DRAGON, Cripto, Endoglin) or intracellular partners (e.g., ARIP1/2), which modulate signaling strength and specificity.

Applications include:

• Bioassays for ligand neutralization or signaling inhibition
• ELISA and binding assays
• Cell culture studies of differentiation, proliferation, or apoptosis
• Mechanistic studies of TGF-β superfamily signaling
• Preclinical models of bone, neural, or reproductive biology

Summary:
Use Recombinant Human Activin RIIA when you need a defined, reproducible tool to probe, block, or quantify activin/BMP/GDF signaling in vitro or in vivo, or to serve as a standard in biochemical assays. Its utility spans developmental biology, disease modeling, and therapeutic research.

Yes, recombinant human Activin RIIA can be used as a standard for quantification and calibration in ELISA assays, though with important considerations regarding the specific formulation and application context.

Formulation Selection for ELISA Standards

The choice of formulation is critical for ELISA applications. Recombinant Activin RIIA is available in two primary formulations:

With Carrier Protein (BSA): This formulation is generally recommended for use as an ELISA standard. The addition of bovine serum albumin enhances protein stability, increases shelf-life, and allows storage at more dilute concentrations, which are all advantageous properties for preparing reliable standard curves.

Carrier-Free: This formulation is recommended when the presence of BSA could interfere with your specific assay design or downstream applications. However, carrier-free proteins require more careful handling to maintain stability.

Preparation and Reconstitution

When using recombinant Activin RIIA as a standard, proper reconstitution is essential. The protein is typically supplied lyophilized and should be reconstituted at 50 µg/mL in sterile phosphate-buffered saline (PBS) containing at least 0.1% human or bovine serum albumin. Upon receipt, store the product immediately at the recommended temperature, and use a manual defrost freezer while avoiding repeated freeze-thaw cycles to maintain protein integrity.

Standard Curve Development

For accurate ELISA calibration, prepare serial dilutions of the reconstituted recombinant protein to establish your standard curve. The dynamic range will depend on your specific assay design and detection method. When preparing standards, ensure consistency in diluent composition, operator technique, pipetting precision, washing procedures, incubation time, and temperature, as variations in these parameters can significantly affect binding and standard curve accuracy.

Important Limitations

While recombinant Activin RIIA can serve as a calibration standard, note that ELISA standards prepared from recombinant proteins are not recommended for bioassay applications where biological activity assessment is required. Additionally, ensure that your assay design accounts for potential cross-reactivity with related proteins in your samples, and validate that the standard curve demonstrates appropriate linearity across your intended measurement range.

Recombinant Human Activin RIIA (ACVR2A) has been validated for several applications in published research, primarily focusing on its role as a signaling modulator and ligand trap in various biological and disease contexts. Key applications include:

1. Bioassay: Used to study the inhibition of activin-induced biological responses, such as mesoderm induction, neural cell differentiation, bone remodeling, hematopoiesis, and regulation of reproductive physiology. For example, recombinant Activin RIIA Fc chimera has been used to inhibit 50% of the biological response due to recombinant human Activin A in cell-based assays.

2. ELISA (Detection): Employed in ELISA formats to detect interactions between Activin RIIA and its ligands or antibodies, such as in functional ELISA studies where immobilized Activin RIIA binds to specific antibodies.

3. Surface Plasmon Resonance (SPR): Used to analyze binding kinetics and affinity between Activin RIIA and its ligands or other interacting proteins.

4. Reporter Gene Assay: Applied in reporter gene assays (e.g., CAGA12-Luc transfected HEK293T/17 cells) to measure activin receptor signaling activity and the effects of inhibitors or neutralizing antibodies.

5. Animal Efficacy Studies: Utilized in preclinical animal models to evaluate the therapeutic potential of Activin RIIA blockade in conditions such as muscle wasting, obesity, and pulmonary arterial hypertension.

6. Neutralization Studies: Used to neutralize activin A production and assess downstream effects, such as reduced parasitemia in disease models.

7. Western Blotting and SDS-PAGE: Employed for protein characterization and detection in biochemical assays.

These applications highlight the versatility of Recombinant Human Activin RIIA in both basic research and therapeutic development, particularly in the context of signaling pathway modulation and disease modeling.

To reconstitute and prepare Recombinant Human Activin RIIA protein for cell culture experiments, dissolve the lyophilized protein in sterile water or phosphate-buffered saline (PBS), typically to a concentration between 50–100 μg/mL, depending on the specific formulation and experimental requirements. Use sterile technique throughout to prevent contamination.

Essential steps and details:

• Reconstitution:

  • For most lyophilized recombinant Activin RIIA proteins, add sterile distilled water or sterile PBS to achieve the desired concentration (commonly 50 μg/mL or 100 μg/mL).
  • If the formulation includes carrier proteins (e.g., BSA), reconstitute in PBS containing at least 0.1% BSA to stabilize the protein.
  • Gently mix by inversion or slow pipetting; avoid vigorous vortexing to prevent protein denaturation.
  • Allow the protein to fully dissolve at room temperature for 10–30 minutes.

• Aliquoting and Storage:

  • After reconstitution, aliquot the solution to avoid repeated freeze-thaw cycles, which can degrade protein activity.
  • Store aliquots at 2–8 °C for short-term use (up to 1 month) or at −20 °C to −70 °C for long-term storage.
  • Avoid multiple freeze-thaw cycles by using single-use aliquots.

• Preparation for Cell Culture:

  • Dilute the reconstituted stock solution into cell culture medium immediately before use to the final working concentration required for your assay (often in the range of 0.03–0.1 μg/mL for bioactivity assays).
  • Ensure the final buffer composition is compatible with your cell culture system (e.g., serum-free or serum-containing media).
  • Filter-sterilize the final working solution if necessary, using a 0.2 μm filter.

Best practices:

• Always consult the product-specific Certificate of Analysis (CoA) or datasheet for recommended reconstitution buffer and concentration, as formulations may vary.
• If the protein is supplied with a tag (e.g., Fc or His), confirm compatibility with your downstream application.
• For sensitive cell types, confirm that the buffer and carrier proteins do not interfere with cell viability or experimental readouts.

Summary Table: Reconstitution Protocols

Always use sterile technique and confirm protein solubility before proceeding with cell culture experiments.