Recombinant Human EDA-A2

Recombinant Human EDA-A2 is used in research to study the specific signaling pathways and biological effects mediated by the EDA-A2/EDA2R axis, which is distinct from the closely related EDA-A1/EDAR pathway. This protein is particularly valuable for investigating mechanisms of muscle degeneration, apoptosis, inflammation, and age-related diseases.

Key scientific applications and rationale include:

• Pathway Specificity: EDA-A2 binds exclusively to the XEDAR (EDA2R) receptor, unlike EDA-A1, which binds EDAR. This receptor specificity allows researchers to dissect the unique roles of EDA-A2/EDA2R signaling in cellular and tissue models.

• Muscle Biology and Disease: Recombinant EDA-A2 has been shown to activate the NF-κB and JNK pathways in skeletal muscle cells, leading to phosphorylation of IκBα and c-Jun, and promoting muscle degeneration. This makes it a critical tool for modeling muscle wasting conditions such as cancer cachexia, Duchenne muscular dystrophy, and sarcopenia.

• Apoptosis and Cell Death: EDA-A2 can induce apoptosis in a p53-dependent manner, as demonstrated by increased pro-apoptotic markers (Bax, cleaved caspase-3) and decreased anti-apoptotic Bcl-2 in treated cells and tissues.

• Inflammation and Ageing: The EDA-A2/EDA2R axis is implicated in inflammatory signaling, immune regulation, and age-related degenerative processes. Recombinant EDA-A2 is used to model these pathways and to explore potential therapeutic interventions for age-associated diseases and chronic inflammation.

• Biomarker and Therapeutic Target Research: EDA2R and its ligand EDA-A2 are emerging as systemic biomarkers for disease vulnerability, biological ageing, and tissue remodeling, making recombinant EDA-A2 essential for biomarker validation and drug discovery studies.

• Bioassays and Functional Studies: Recombinant EDA-A2 is used in bioassays to characterize receptor binding, downstream signaling, and functional cellular responses, including surface plasmon resonance (SPR) and cell-based assays.

In summary, using recombinant human EDA-A2 enables precise investigation of EDA2R-mediated signaling and its roles in muscle biology, apoptosis, inflammation, ageing, and disease, supporting both basic research and translational applications.

Yes, recombinant human EDA-A2 can be used as a standard for quantification and calibration in ELISA assays. This protein is specifically suitable for this application, particularly when you need to quantify EDA-A2 in biological samples such as plasma, serum, or cell culture supernatants.

Preparation and Reconstitution

The recombinant EDA-A2 protein is typically supplied in lyophilized form and requires reconstitution before use. Standard reconstitution involves dissolving the protein at 100 μg/mL in sterile PBS (phosphate-buffered saline). For cell or tissue culture applications and ELISA standard preparation, the protein formulation containing BSA (bovine serum albumin) as a carrier protein is recommended, as it provides enhanced stability during handling and storage.

Standard Curve Development

When preparing your ELISA standard curve, you should establish a concentration range appropriate for your target analyte levels. Typical standard curves range from 0 to 1000 pg/mL, though this can extend to 3000 pg/mL if your predicted protein concentrations are extremely high. For EDA-A2 specifically, assay ranges of 10-2500 pg/mL are commonly employed in sandwich ELISA formats.

Storage and Stability Considerations

To maintain the integrity of your recombinant EDA-A2 standard, store it in a manual defrost freezer and avoid repeated freeze-thaw cycles. The protein is shipped at ambient temperature, so immediate storage at the recommended temperature upon receipt is essential for preserving its functionality in binding activity assays and maintaining reproducibility across multiple assay runs.

The recombinant protein will provide reliable quantification when used with appropriate curve-fitting software, such as 4-parameter logistic (4-PL) or 5-parameter logistic (5-PL) regression models, which are well-suited for most ELISA calibration curves.

Recombinant Human EDA-A2 has been validated for several applications in published research, primarily in the context of functional bioassays, binding activity assays, and mechanistic studies of signaling pathways.

Key validated applications include:

• Bioassays: Recombinant EDA-A2 has been used to study its biological activity, such as its ability to activate downstream signaling through its receptor EDA2R/XEDAR. For example, it has been applied to investigate muscle atrophy in cancer cachexia models and to study p53-dependent regulation in chemotherapy-induced hair loss.
• Binding Activity Assays: The protein has been validated for binding activity, confirming its interaction with its specific receptor (EDA2R/XEDAR).
• Surface Plasmon Resonance (SPR): Used to characterize the binding kinetics and affinity between EDA-A2 and its receptor.
• Cellular Signaling Studies: Recombinant EDA-A2 has been used to activate and study signaling pathways such as NF-κB and JNK in cell-based assays, including phosphorylation of IκBα and c-Jun, and to assess downstream effects like apoptosis and muscle degeneration.
• Lipid Production Assays: It has been used to demonstrate increased lipid production in EDA2R-expressing human sebocytes.
• ELISA Standard: While not always explicitly stated in publications, recombinant EDA-A2 is commonly used as a standard or positive control in ELISA-based quantification of EDA-A2 levels.

Supporting details and specific research contexts:

• Muscle Atrophy and Degeneration: Recombinant EDA-A2 was used in mouse models and human skeletal muscle cells to show that EDA-A2/EDA2R signaling promotes muscle degeneration, particularly through IκBα phosphorylation and NF-κB pathway activation.
• Hair Follicle and Apoptosis Studies: In cultured human hair follicles, EDA-A2 treatment led to increased pro-apoptotic markers and decreased anti-apoptotic proteins, supporting its use in apoptosis-related bioassays.
• Metabolic and Obesity Research: EDA-A2 administration in mice increased JNK phosphorylation and affected glucose metabolism, validating its use in metabolic pathway studies.
• Sebocyte Function: EDA-A2 increased lipid production in EDA2R-expressing sebocytes, demonstrating its utility in functional assays related to skin biology.

Summary Table of Validated Applications

These applications are supported by peer-reviewed studies and product validation data, confirming the utility of recombinant human EDA-A2 in diverse experimental systems relevant to cell signaling, muscle biology, skin biology, and metabolic research.

Reconstitution Protocol

Recombinant Human EDA-A2 protein is supplied in lyophilized form and requires proper reconstitution before use in cell culture experiments. The reconstitution process is straightforward but requires attention to detail to maintain protein integrity and biological activity.

Initial Preparation

Begin by centrifuging the vial at 5,000×g for 5 minutes to concentrate the lyophilized powder at the bottom of the tube. This ensures you are adding the reconstitution buffer to all the protein material.

Reconstitution Buffer and Concentration

For the standard formulation containing bovine serum albumin (BSA) as a carrier protein, reconstitute the protein at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin. If using the carrier-free version, reconstitute at 100 μg/mL in sterile PBS without additional carrier protein.

The carrier protein is particularly important for cell culture applications, as it enhances protein stability, increases shelf-life, and allows storage at more dilute concentrations. The presence of BSA does not interfere with typical cell culture experiments.

Reconstitution Procedure

Add the appropriate volume of sterile reconstitution buffer to the vial. Allow the vial to reconstitute for 15-30 minutes at room temperature with gentle agitation. Avoid vigorous shaking, which can cause foaming and potentially denature the protein. Gentle swirling of the vial is typically sufficient for complete dissolution.

Storage and Stability

Short-term Storage

After reconstitution under sterile conditions, the protein may be stored at 2-8°C for up to 1 month.

Long-term Storage

For extended storage, maintain the reconstituted protein at -20 to -70°C for up to 3 months. The lyophilized protein itself is stable for 6-12 months when stored desiccated at -20 to -70°C.

Critical Storage Considerations

Use a manual defrost freezer and avoid repeated freeze-thaw cycles, as these can compromise protein stability and biological activity. If you require multiple aliquots, consider preparing smaller working volumes to minimize freeze-thaw exposure.

Quality Specifications

The recombinant protein is typically supplied at 95% purity as determined by SDS-PAGE under reducing conditions and has a molecular weight of approximately 24.7 kDa. The protein is derived from mammalian cell expression systems, ensuring proper post-translational modifications necessary for biological activity.