Recombinant Human Activin RIIB

Recombinant Human Activin RIIB is widely used in research because it is a high-affinity receptor for activins and related ligands, enabling precise modulation and study of the activin signaling pathway, which is crucial in diverse biological processes such as cell differentiation, muscle growth, hematopoiesis, and disease modeling.

Key reasons to use Recombinant Human Activin RIIB in research applications:

• High Ligand Affinity: Activin RIIB displays a 3- to 4-fold higher affinity for activin A compared to other type II receptors, making it especially effective for ligand-binding studies and as a decoy receptor to sequester activins in functional assays.
• Pathway Modulation: As a soluble recombinant protein, Activin RIIB can act as a potent antagonist of activin signaling by binding and neutralizing activin ligands, thereby blocking downstream signaling events. This is valuable for dissecting the roles of activin and related TGF-β family members in cellular models.
• Functional Assays: Recombinant Activin RIIB is used to inhibit activin-induced biological effects, such as hemoglobin expression in K562 cells, providing a robust tool for validating pathway specificity and for screening modulators of activin signaling.
• Therapeutic Research: The activin pathway is implicated in muscle wasting, anemia, fibrosis, and cancer. Recombinant Activin RIIB is used to model and potentially counteract these conditions in preclinical studies, including the development of ligand traps and therapeutic fusion proteins.
• Protein-Protein Interaction Studies: Recombinant Activin RIIB is suitable for ELISA, SPR, and other binding assays to quantify interactions with activins, BMPs, and related ligands, supporting drug discovery and mechanistic research.
• High Purity and Reproducibility: Recombinant preparations are typically >95% pure and endotoxin-free, ensuring consistent results in sensitive cell-based and biochemical assays.

Applications include:

• Blocking or neutralizing activin signaling in cell culture or animal models.
• Studying receptor-ligand interactions and downstream signaling mechanisms.
• Screening for small molecules or biologics that modulate the activin pathway.
• Investigating the role of activin signaling in development, disease, and tissue homeostasis.

In summary, Recombinant Human Activin RIIB is a versatile and essential reagent for research on TGF-β superfamily signaling, enabling both mechanistic studies and translational research targeting activin-related pathways.

Recombinant Human Activin RIIB can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity, properly formulated, and validated for your specific assay system. Several recombinant forms of Activin RIIB are explicitly recommended for use as ELISA standards in research applications.

Key considerations for use as an ELISA standard:

• Purity and Formulation: The recombinant protein should be highly pure (typically >90–95%) and free of contaminants that could interfere with the assay. Proteins formulated with stabilizers such as BSA are often preferred for ELISA standards due to improved stability and reproducibility.
• Validation: It is essential to validate that the recombinant Activin RIIB behaves similarly to the native protein in your ELISA system. This includes confirming parallelism of standard curves and consistent detection across dilutions.
• Calibration: Prepare a standard curve using serial dilutions of the recombinant protein in the same buffer or diluent as your samples to ensure accurate quantification. Follow best practices for standard preparation, including careful pipetting and mixing to avoid technical errors.
• Controls: Include appropriate positive and negative controls in your assay to confirm specificity and accuracy of quantification.

Limitations and best practices:

• Research Use Only: Most recombinant standards are intended for research use and not for diagnostic procedures.
• Batch Consistency: Use the same lot of recombinant protein for all standards in a given experiment to minimize variability.
• Documentation: Refer to the Certificate of Analysis and product datasheet for specific instructions on reconstitution, storage, and handling.

Summary Table: Recombinant Activin RIIB as ELISA Standard

In conclusion, recombinant Human Activin RIIB is suitable as an ELISA standard if it meets purity, formulation, and validation criteria for your assay system.

Recombinant Human Activin RIIB has been validated in published research for several key applications, primarily as a functional ligand trap and in cell-based assays to study TGF-β superfamily signaling, muscle growth regulation, and metabolic processes.

Key validated applications include:

• Inhibition of Activin A-induced hemoglobin expression in K562 cells: This is the most widely cited functional assay, where recombinant Activin RIIB is used to block Activin A signaling, resulting in reduced hemoglobin expression in the K562 human chronic myelogenous leukemia cell line. This assay is a standard for validating the biological activity of recombinant Activin RIIB proteins.

• Protein-protein interaction studies: Recombinant Activin RIIB is used in ELISA and binding assays to quantify its interaction with ligands such as Activin A and GDF-8 (myostatin), as well as to determine binding affinities and inhibitory concentrations (e.g., EC50, IC50). These assays are critical for characterizing ligand-receptor specificity and potency.

• Cell analysis and cell-based functional assays: The protein is employed in various cell analysis methods to study signaling pathways, receptor-ligand interactions, and downstream effects on gene expression and cellular phenotype.

• Therapeutic development and metabolic research: Recombinant Activin RIIB has been used as a research tool in preclinical studies and drug development, particularly for investigating its role in muscle mass regulation, obesity, and metabolic diseases. It serves as a model for ligand trap therapies and as a comparator in studies of monoclonal antibodies targeting the Activin pathway.

• Immunological and cell biology research: The protein is utilized in broader immunological and cell biology studies, including analyses of TGF-β superfamily signaling, muscle growth, and fat metabolism.

Summary table of validated applications:

These applications are supported by multiple published studies and product validation data, establishing recombinant human Activin RIIB as a versatile tool in both basic and translational research.

Reconstitution Protocol

Pre-reconstitution preparation is critical for successful protein recovery. Before opening the vial, centrifuge it in a microcentrifuge for 20-30 seconds to drive any protein that may be lodged in the cap or on the sides to the bottom of the vial. Additionally, allow the lyophilized powder to warm to room temperature before opening to improve solubility.

Reconstitution Conditions

Solvent selection depends on the specific product formulation. Most Activin RIIB proteins can be reconstituted with sterile, distilled water, though some formulations may require alternative buffers such as phosphate-buffered saline (PBS). Always consult the Certificate of Analysis (CoA) or product data sheet for the exact diluent recommended for your specific preparation.

Concentration recommendations typically range from 0.1 to 1.0 mg/mL, with some formulations recommending higher concentrations around 100-200 μg/mL depending on the expression system and tag configuration. For example, if reconstituting 100 μg of protein, add between 100 μL and 1 mL of the appropriate solvent to achieve the desired concentration.

Handling during reconstitution is essential to maintain protein integrity. Avoid vortexing or vigorously pipetting the protein solution, as this can cause denaturation and aggregation. If solubility issues arise, allowing the reconstituted protein to incubate overnight at 4°C may help resolve them.

Storage of Reconstituted Protein

Short-term storage of reconstituted working aliquots should be maintained at -20°C to -80°C for extended periods. Ensure that working aliquots contain at least 10 μL of protein solution with carrier protein when applicable. Alternatively, short-term storage at 2-8°C is permissible for up to one month.

Critical consideration: Avoid multiple freeze-thaw cycles, as repeated freezing significantly affects the pH of the protein solution and can cause protein denaturation. Prepare aliquots of appropriate size to minimize the need for repeated thawing.

Verification of Reconstitution

After reconstitution, confirm the presence of product protein by running a small amount on SDS-PAGE analysis. A protein band at the expected molecular weight should be visible with as little as 10 ng of protein loaded on an acrylamide gel. This verification step ensures complete recovery and proper reconstitution of your protein preparation.