Recombinant Mouse EphA7

Recombinant Mouse EphA7 is a valuable tool for research applications focused on cell differentiation, neural development, tissue regeneration, and cellular signaling due to its role as a receptor tyrosine kinase involved in key developmental and repair processes.

Key scientific reasons to use Recombinant Mouse EphA7:

• Promotes Myogenic Differentiation: EphA7 is critical for the conversion of myoblasts to terminally differentiated myocytes during skeletal muscle development and regeneration. Exogenous recombinant EphA7 can accelerate differentiation in vitro, rescue differentiation defects in EphA7-deficient cells, and enhance commitment to differentiation in wild-type cells.

• Neural Development and Synaptic Stabilization: EphA7 is required for the stabilization of synaptic terminals, particularly in parvalbumin-positive basket cells in the hippocampus. Overexpression of EphA7 increases gephyrin clustering, indicating its sufficiency in promoting synaptic organization and potentially influencing learning and memory processes.

• Cell-Cell Communication and Tissue Patterning: EphA7 participates in juxtacrine signaling, influencing cell positioning, adhesion, and migration. It is involved in the ephrin receptor and MAPK signaling pathways, which are essential for tissue architecture and developmental patterning.

• Regulation of Proliferation and Tumor Suppression: EphA7 has been characterized as a tumor suppressor in several cancers and acts as a negative regulator of cell proliferation through reverse signaling mechanisms.

• Cellular Reprogramming: Truncated forms of EphA7 can promote somatic cell reprogramming by modulating ERK1/2 activity, suggesting utility in studies of cell fate and regenerative medicine.

Typical research applications include:

• In vitro differentiation assays for muscle or neural cells.
• Functional studies of cell signaling pathways (e.g., MAPK, ephrin receptor signaling).
• Investigation of tissue regeneration and repair mechanisms.
• Analysis of synaptic development and plasticity in neural tissue.
• Cancer biology and studies of tumor suppressor mechanisms.

Best practices:

• Use recombinant EphA7 in controlled in vitro systems to dissect its role in differentiation and signaling.
• Employ appropriate concentrations and formats (e.g., Fc chimeras, extracellular domains) to mimic physiological signaling conditions.
• Validate biological activity using kinase assays or functional readouts relevant to your cell type and experimental design.

In summary, recombinant Mouse EphA7 enables precise manipulation of EphA7-mediated signaling, facilitating mechanistic studies in muscle differentiation, neural development, tissue patterning, and disease models where EphA7 function is implicated.

You can use recombinant mouse EphA7 as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is of high purity, its concentration is accurately determined, and it is immunologically recognized by the antibodies used in your assay.

Key considerations:

• Purity and Form: The recombinant EphA7 should be highly purified (typically >95% by SDS-PAGE) and ideally in a form that matches the native protein or the epitope recognized by your ELISA antibodies.
• Concentration Determination: The protein concentration must be accurately measured, as errors here will directly affect your standard curve and quantification accuracy.
• Immunoreactivity: The recombinant protein must be recognized by the capture and detection antibodies in your ELISA. If your ELISA is designed for full-length EphA7 but your recombinant standard is a fragment, ensure the fragment contains the relevant epitopes.
• Standard Curve Preparation: Prepare a fresh standard curve for each assay run using serial dilutions of the recombinant protein. Do not rely on demonstration curves from kit datasheets; always generate your own under your assay conditions.
• Lot-to-Lot Variability: Recombinant proteins may show variability in immunoreactivity between lots. It is best practice to assign the value of your standard based on its performance in the ELISA, not just the mass on the vial label.
• Validation: If using a recombinant standard not supplied with a commercial kit, validate its performance by spike-and-recovery experiments and parallelism testing to ensure it behaves similarly to endogenous EphA7 in your sample matrix.

Limitations:

• If your recombinant EphA7 is a fragment or fusion protein (e.g., with tags), confirm that these modifications do not interfere with antibody binding or assay performance.
• If your ELISA kit is calibrated with a different standard (e.g., a different isoform or full-length protein), results may not be directly comparable unless validated.

Best Practices:

• Use the recombinant protein to generate a standard curve within the validated range of your assay.
• Assign the standard’s value based on its measured performance in the ELISA, not solely on the manufacturer’s stated concentration.
• Validate the standard by comparing its recovery and parallelism with endogenous EphA7 in your sample type.

In summary, recombinant mouse EphA7 can be used as a standard in ELISA quantification, but careful validation and standard curve preparation are essential for accurate and reliable results.

Recombinant Mouse EphA7 has been validated for several key applications in published research, primarily in kinase activity assays, cell signaling studies, and SDS-PAGE analysis. It is also used in functional studies related to neural development, immune regulation, and cancer biology.

Validated Applications:

• Kinase Activity Assays: Recombinant Mouse EphA7 has demonstrated biological activity in kinase assays, specifically using Poly E4Y peptide as a substrate, with a reported specific activity of 35 nmol/min/mg. This confirms its utility in enzymatic and phosphorylation studies.

• SDS-PAGE: The protein has been analyzed by SDS-PAGE to confirm purity and molecular weight (~73 kDa), supporting its use in protein characterization and quality control.

• Cell Signaling Studies: EphA7 is involved in the MAPK/ERK signaling pathway. Research has shown that EphA7 mediates ERK1/2 phosphorylation, and its suppression leads to reduced ERK activation in leukemic cell models. This validates its use in studies of receptor tyrosine kinase signaling and downstream pathway analysis.

• Functional Studies: Recombinant EphA7 has been used to investigate its role in neural development (axon guidance, cortical mapping), immune cell interactions, and tissue architecture. It is also implicated in caspase-dependent proapoptotic activity and cell adhesion/repulsion mechanisms.

• Cellular Reprogramming: Truncated forms of EphA7 have been shown to promote somatic cell reprogramming by reducing ERK1/2 activity, indicating its application in stem cell and developmental biology research.

Additional Context:

• Biomarker Research: EphA7 has been studied as a biomarker in cancer, particularly in relation to immune checkpoint inhibitor response and methylation status in cervical cancer. While these studies focus on endogenous EphA7, recombinant protein may be used for assay development or mechanistic studies.

• Protein-Protein Interaction Studies: EphA7 interacts with ephrin-A ligands (notably EFNA5), making it suitable for binding assays and studies of receptor-ligand dynamics.

Summary Table of Validated Applications

These applications are supported by published research and supplier validation data, confirming the recombinant protein's utility in diverse experimental settings.

To reconstitute and prepare Recombinant Mouse EphA7 protein for cell culture experiments, follow these general best practices based on protocols for similar recombinant proteins:

1. Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
2. Reconstitute the protein in sterile, deionized water or sterile PBS, depending on the formulation and intended downstream application. A typical concentration range is 0.1–1.0 mg/mL. For most cell culture applications, starting at 0.1–0.5 mg/mL is appropriate.
3. Gently mix by swirling or gentle pipetting. Avoid vortexing or vigorous pipetting, as this can denature the protein.
4. Allow the protein to dissolve at room temperature for 15–30 minutes with gentle agitation.
5. If required, add a carrier protein (such as 0.1% BSA or 5% HSA) to stabilize the solution, especially for long-term storage or if the protein will be used at low concentrations.
6. Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can degrade protein activity.
7. Storage: After reconstitution, store aliquots at 2–8 °C for short-term use (up to 1 week) or at –20 °C to –70 °C for long-term storage. Avoid repeated freeze-thaw cycles.

Preparation for cell culture:

• Before adding to cells, dilute the reconstituted protein to the desired working concentration using sterile cell culture medium or buffer compatible with your assay.
• Filter-sterilize the final solution if sterility is required and the protein is not already in a sterile buffer.

Additional notes:

• Always consult the specific product datasheet for formulation details and recommended reconstitution buffer, as some preparations may contain stabilizers or require specific pH conditions.
• If the protein is supplied in a buffer containing glycerol or other additives, it may be ready-to-use and not require reconstitution.

Summary of key steps:

• Centrifuge vial → Add sterile water or PBS (0.1–1.0 mg/mL) → Gentle mixing → Incubate at RT 15–30 min → Add carrier if needed → Aliquot and store appropriately → Dilute for cell culture use.

These steps are consistent with standard protocols for recombinant EphA7 and similar proteins. Always verify with the specific product documentation for any unique requirements.