Recombinant Human EDAR

Recombinant Human EDAR is a valuable tool for research applications due to its critical role in key signaling pathways and its relevance to both developmental biology and disease mechanisms. Here are several reasons why you should consider using Recombinant Human EDAR in your research:

1. Study of Signaling Pathways

EDAR is a receptor for Ectodysplasin-A1 (EDA-A1) and activates the NF-κB and JNK signaling pathways upon ligand binding. Using recombinant EDAR allows you to:

• Investigate the molecular mechanisms of these signaling cascades in vitro.
• Examine how EDAR activation influences downstream gene expression and cellular responses.

2. Modeling Human Disease

Mutations in EDAR are associated with hypohidrotic ectodermal dysplasia (HED), a genetic disorder affecting skin, hair, teeth, and sweat glands. Recombinant EDAR enables:

• Functional studies of disease-causing mutations.
• Assessment of how specific variants affect receptor activity and signaling.

3. Drug Discovery and Target Validation

Recombinant EDAR can be used in high-throughput screening assays to:

• Identify small molecules or biologics that modulate EDAR activity.
• Validate EDAR as a potential therapeutic target for conditions such as ectodermal dysplasias or even cancer, given its tumor suppressor role in melanoma.

4. Protein-Protein Interaction Studies

Recombinant EDAR facilitates the study of interactions with:

• Its ligand EDA-A1.
• Downstream signaling proteins (e.g., EDARADD).
• Other components of the NF-κB and JNK pathways.

5. Structural Biology

High-purity recombinant EDAR is essential for:

• Structural studies (e.g., X-ray crystallography, cryo-EM) to determine the receptor’s conformation and ligand-binding interfaces.
• Mapping epitopes for antibody development.

6. Diagnostic Assays

Recombinant EDAR can serve as:

• A positive control in immunoassays (e.g., ELISA, Western blot).
• An antigen for antibody production and validation.

7. Functional Assays

You can use recombinant EDAR to:

• Develop cell-based assays to measure receptor activation and downstream signaling.
• Test the effects of potential therapeutic agents on EDAR-mediated responses.

8. Consistency and Reproducibility

Recombinant proteins offer high batch-to-batch consistency, ensuring reliable and reproducible results across experiments.

In summary, Recombinant Human EDAR is a versatile and essential reagent for studying signaling pathways, modeling disease, drug discovery, protein interactions, structural biology, and diagnostic development. Its use can significantly enhance the quality and impact of your research.

Yes, Recombinant Human EDAR can be used as a standard for quantification or calibration in ELISA assays, provided that the recombinant protein is well-characterized, purified, and its concentration is accurately determined.

Key Points Supporting the Use of Recombinant Human EDAR as a Standard:

1. Purpose of Recombinant Standards in ELISA:

   • Recombinant proteins are commonly used to generate standard curves in ELISA assays for quantifying target analytes, especially when purified native proteins are not available or are difficult to obtain.
   • As noted in several sources, recombinant proteins are suitable for preparing standard curves if they are purified and their concentration is measured accurately (e.g., by HPLC or spectrophotometry) .

2. Commercial ELISA Kits for EDAR:

   • Many commercial Human EDAR ELISA kits (e.g., MyBioSource, BosterBio, OriGene, RayBiotech) explicitly state that they include recombinant Human EDAR as the standard for calibration and quantification [3, 7, 11, 13].
   • These kits are designed to detect both natural and recombinant forms of EDAR, confirming that recombinant EDAR is a valid and recognized standard for these assays [1, 3, 5, 7, 11].

3. Calibration and Quantification:

   • The standard curve in ELISA is typically prepared using serial dilutions of the recombinant protein, and sample concentrations are interpolated from this curve .
   • For accurate quantification, it is important to ensure that the recombinant standard is calibrated against a reference standard (e.g., NIBSC or WHO standards) if available, to ensure harmonization and comparability across assays .

4. Validation and Matrix Effects:

   • When using recombinant EDAR as a standard, it is important to validate the assay for accuracy, precision, and linearity in the sample matrix of interest (e.g., serum, plasma, cell culture supernatant) [4, 8].
   • Spike/recovery and dilution linearity experiments should be performed to confirm that the recombinant standard behaves similarly to the endogenous analyte in the sample matrix .

Recommendations:

• Use a highly purified recombinant Human EDAR with a known concentration.
• Prepare the standard curve using serial dilutions of the recombinant protein in the same buffer or matrix as your samples.
• Validate the assay for accuracy, precision, and linearity in your specific sample type.
• If possible, calibrate your recombinant standard against a reference standard for improved comparability.

In summary, Recombinant Human EDAR is a suitable and widely used standard for quantification and calibration in ELISA assays for EDAR, as long as it is properly characterized and validated for your specific application.

Recombinant Human EDAR has been validated in published research for several key applications, primarily in studies of signaling, disease modeling, and functional assays.

Validated Applications in Published Research:

• Functional and Signaling Assays: Recombinant human EDAR has been used to study its role as a receptor in the ectodysplasin signaling pathway, particularly in the context of hair, tooth, and gland development. For example, mouse models expressing human EDAR variants have been used to assess phenotypic changes and signaling potency, including the impact of specific mutations such as V370A on NF-κB signaling and ectodermal appendage development.

• Disease Modeling: Recombinant EDAR has been utilized in generating mouse models to mimic human genetic variants, allowing researchers to study the consequences of EDAR mutations associated with ectodermal dysplasias and to explore evolutionary adaptations in human populations.

• Tumor Suppression Studies: EDAR has been validated in cancer research, specifically in melanoma models. Recombinant EDAR expression constructs were used in xenograft mouse models to demonstrate its tumor suppressor function, with intra-tumoral delivery of EDAR shown to inhibit tumor growth. These studies involved both in vivo (animal) and in vitro (cell culture) systems, with downstream analysis by quantitative RT-PCR and other molecular assays.

• Protein Interaction and Binding Assays: Recombinant EDAR has been used in binding activity assays and bioassays to characterize its interactions with ligands such as EDA-A1 and EDA-A2, and to assess downstream signaling events.

• Western Blot and ELISA: Some commercial sources and research protocols report the use of recombinant human EDAR as a standard or control in Western blot and ELISA applications, although direct citations from primary research literature are limited.

Summary Table:

Key Context:

• Most published research uses recombinant human EDAR in in vivo mouse models or in vitro cell-based assays to study developmental biology, disease mechanisms, and receptor signaling.
• Applications in Western blot and ELISA are more commonly reported in product datasheets and secondary literature, with primary research focusing on functional and mechanistic studies.

If you need details on protocols or specific experimental setups for any of these applications, please specify the context or research focus.

To reconstitute and prepare Recombinant Human EDAR protein for cell culture experiments, first confirm whether your protein is supplied lyophilized and whether it contains carrier proteins such as BSA, as this affects the reconstitution protocol.

General Reconstitution Protocol:

• Carrier-Free (no BSA):

  • Reconstitute the lyophilized protein at a concentration of 100 μg/mL in sterile PBS (phosphate-buffered saline).
  • Gently mix by swirling or inverting; avoid vigorous shaking or vortexing to prevent protein denaturation or foaming.
  • Allow the vial to sit at room temperature for 15–30 minutes to ensure complete dissolution.
  • If the protein is difficult to dissolve, gentle pipetting (not vortexing) or brief sonication in a water bath can help, but avoid introducing bubbles.

• With Carrier (contains BSA):

  • Reconstitute at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin to stabilize the protein.
  • Follow the same gentle mixing and incubation steps as above.

Additional Best Practices:

• Sterility: Always use sterile reagents and equipment to prevent contamination in cell culture applications.
• Aliquoting: After reconstitution, aliquot the protein into single-use portions to avoid repeated freeze-thaw cycles, which can degrade protein activity.
• Storage: Store lyophilized protein at –20°C to –70°C (desiccated). After reconstitution, store aliquots at 4°C for short-term use (2–7 days) or at –20°C for longer-term storage. Avoid repeated freeze-thaw cycles.
• Concentration Adjustment: If a different working concentration is needed, dilute the reconstituted stock with sterile PBS or cell culture medium immediately before use.
• Handling: Centrifuge the vial briefly before opening to collect all material at the bottom. Do not mix by vortexing or harsh pipetting.

Application in Cell Culture:

• Before adding to cells, ensure the final buffer composition is compatible with your cell culture system (e.g., isotonic, pH 7.2–7.4, free of toxic preservatives).
• If using in sensitive assays, consider using carrier-free protein to avoid interference from BSA or other additives.

Summary Table:

Always consult the specific product datasheet for any unique requirements or recommendations for your recombinant EDAR protein batch.