Recombinant Rat Agrin

Recombinant Rat Agrin is widely used in research due to its critical roles in neuromuscular junction (NMJ) formation, tissue regeneration, and cell signaling, making it a valuable tool for studies in neuroscience, regenerative medicine, and cell biology.

Key reasons to use recombinant rat agrin in research applications:

• NMJ Formation and Synaptic Biology: Agrin is essential for the aggregation of acetylcholine receptors (AChRs) at the NMJ, a process fundamental to synapse formation and maintenance in skeletal muscle. Application of recombinant agrin to muscle fibers induces robust, long-lasting AChR clustering, enabling detailed studies of synaptogenesis and neuromuscular diseases.

• Tissue Repair and Regeneration: Agrin has pleiotropic effects on tissue repair, promoting proliferation and regeneration in cardiac, neural, and musculoskeletal tissues. Recombinant agrin has been shown to enhance cardiac regeneration after myocardial infarction, support osteochondral (bone and cartilage) repair, and improve wound healing by modulating extracellular matrix (ECM) mechanics and cell migration.

• Cell Signaling and Mechanotransduction: Agrin modulates cell signaling pathways, including those involved in mechanoperception, cytoskeletal organization, and cell motility, particularly in keratinocytes and other epithelial cells. This makes it useful for dissecting ECM-cell interactions and mechanobiology.

• Immunological and Neurodegenerative Research: Agrin participates in T cell activation and immunological synapse formation, and is implicated in neurodegenerative conditions such as Alzheimer’s disease, where it interacts with amyloid-beta peptides. Recombinant agrin can thus be used to model these processes in vitro.

• Validated Bioactivity: Recombinant rat agrin is validated for bioactivity in various cell-based assays, including induction of AChR clustering in myotubes, activation of MuSK phosphorylation, and heparin binding, ensuring reproducibility and reliability in experimental systems.

• Species Relevance: Using rat agrin is particularly appropriate for studies involving rat primary cells, tissues, or in vivo models, ensuring species-specific interactions and physiological relevance.

In summary, recombinant rat agrin is a versatile reagent for investigating synaptic development, tissue regeneration, ECM biology, and disease mechanisms, with robust validation in multiple research contexts.

Recombinant Rat Agrin can be used as a standard for quantification or calibration in ELISA assays, provided it is compatible with your assay’s antibody pair and format. Several sources recommend recombinant Agrin (especially with BSA as a carrier) for use as an ELISA standard. However, there are important technical considerations:

• Formulation: Recombinant Agrin with BSA is generally advised for use as an ELISA standard due to enhanced stability. Carrier-free versions are available if BSA interferes with your assay.
• Antibody Recognition: The recombinant Agrin must contain the same epitopes as the native protein recognized by the capture and detection antibodies in your ELISA. Most commercial ELISA kits are validated with their own standards, which may be full-length or specific fragments of Agrin. If your recombinant protein differs in sequence, post-translational modifications, or domain structure, antibody binding and quantification accuracy may be affected.
• Calibration and Quantification: ELISA kits are typically calibrated using a master calibrator, and the standards provided are validated for consistent performance. If you substitute the kit standard with your recombinant Agrin, you must validate that the standard curve generated is accurate and reproducible. Differences in immunoreactivity or protein quantification methods (e.g., mass by absorbance vs. immunoreactivity) can lead to discrepancies in measured concentrations.
• Best Practices: If using recombinant Agrin as a standard:
  • Prepare a standard curve using serial dilutions of the recombinant protein.
  • Confirm that the standard curve is linear and covers the expected range of your samples.
  • Validate recovery and parallelism by spiking known amounts of recombinant Agrin into sample matrices.
  • Assign the value of your standard based on its performance in the ELISA, not solely on the mass stated on the vial.

Summary Table: Key Considerations for Using Recombinant Agrin as an ELISA Standard

Conclusion:
You can use recombinant Rat Agrin as a standard for ELISA quantification, but you must validate its performance in your specific assay to ensure accurate and reliable results. Always check that the recombinant protein matches the immunological properties required by your ELISA antibodies and format.

Recombinant Rat Agrin has been validated in published research for several key applications, primarily focused on its bioactivity in cell culture and functional assays related to neuromuscular junction (NMJ) formation, cell proliferation, migration, and synaptogenesis.

Validated Applications in Published Research:

• Bioassays for Acetylcholine Receptor (AChR) Clustering:
  Recombinant rat Agrin is widely used to induce AChR clustering in myotubes (e.g., C2C12, chick myotubes), a hallmark assay for NMJ formation and function. This activity is often quantified by immunofluorescence or Western blot for MuSK phosphorylation.

• Cell Proliferation and Regeneration Assays:
  In cardiac research, recombinant rat Agrin has been shown to promote proliferation of cardiomyocytes derived from mouse and human iPSCs, validated by markers such as Ki67, phospho-histone H3, and aurora kinase B.

• Cell Migration and Invasion Assays:
  Soluble recombinant Agrin has been used to rescue migration and invasion defects in Agrin-depleted cells, especially in cancer models (e.g., hepatocellular carcinoma), assessed by wound scratch, transwell migration, and invasion assays.

• Synaptogenesis and Neural Repair:
  Agrin is implicated in synaptogenesis and neural repair after stroke, with studies using recombinant protein to investigate its role in synaptic formation and plasticity in vivo and in vitro.

• Immunohistochemistry:
  Recombinant Agrin has been used as a positive control or antigen in immunohistochemical studies of tissue sections, particularly in liver and neural tissues.

• Western Blot and ELISA:
  Recombinant rat Agrin is validated for use as a standard or antigen in Western blot and ELISA protocols, typically at concentrations of 1–2 µg/mL for Western blot and 0.5–1.0 µg/mL for ELISA.

• Cell Attachment and ECM Signaling Assays:
  Agrin’s role as an extracellular matrix (ECM) sensor has been studied using recombinant protein to assess focal adhesion integrity, cell attachment, and actin polymerization, often in combination with fibronectin.

Summary Table of Validated Applications

Additional Notes:

• Most published studies use recombinant rat Agrin in in vitro cell culture systems, but some extend to in vivo models for neural repair, cardiac regeneration, and cancer biology.
• The protein is typically used at nanomolar to low micromolar concentrations, depending on the assay.
• Validation includes functional readouts (e.g., receptor clustering, cell proliferation) and molecular assays (e.g., Western blot, ELISA).

If you require protocols or specific concentrations for a particular application, please specify the intended use.

To reconstitute and prepare Recombinant Rat Agrin protein for cell culture experiments, dissolve the lyophilized protein in sterile PBS (phosphate-buffered saline), typically at a concentration of 100–500 μg/mL, and include at least 0.1% carrier protein such as bovine serum albumin (BSA) or human serum albumin (HSA) to stabilize the protein and prevent adsorption to surfaces.

Step-by-step protocol:

1. Equilibrate materials: Allow the lyophilized protein vial and reconstitution buffer (sterile PBS, pH 7.2–7.4) to reach room temperature before opening.

2. Centrifuge vial: Briefly centrifuge the vial (3000–3500 rpm, 5 min) to collect the powder at the bottom and minimize loss when opening.

3. Add buffer:

   • For formulations with carrier protein: Add sterile PBS containing at least 0.1% BSA or HSA to achieve a final concentration of 500 μg/mL.
   • For carrier-free formulations: Add sterile PBS to achieve a final concentration of 100 μg/mL.

4. Dissolve gently:

   • Do not vortex. Gently mix by pipetting up and down or by slow inversion. Let the vial sit at room temperature for 15–30 min, gently agitating occasionally until fully dissolved.
   • If undissolved particles remain, continue gentle mixing for up to 2 hours at room temperature.

5. Aliquot and storage:

   • Prepare aliquots sized for single-use experiments (≥20 μL) to avoid repeated freeze-thaw cycles.
   • Store aliquots at –20°C to –80°C for long-term storage, or at 2–8°C for short-term use (up to 1 week).
   • Avoid repeated freeze-thaw cycles, as this can reduce protein activity.

6. Dilution for cell culture:

   • Dilute the reconstituted stock solution into cell culture medium or PBS containing carrier protein (e.g., 0.1% BSA or 10% FBS) to the desired working concentration for your experiment.

Additional notes:

• Always consult the specific datasheet for your Agrin protein batch for recommended buffer and concentration, as formulations may differ (e.g., presence or absence of carrier protein, trehalose, etc.).
• For sensitive applications, filter-sterilize the final solution using a 0.2 μm filter if required.
• Agrin is a large, glycosylated extracellular matrix protein; gentle handling is essential to preserve its biological activity.

Summary of key points:

• Buffer: Sterile PBS (pH 7.2–7.4), with 0.1% BSA/HSA if possible.
• Concentration: 100–500 μg/mL for stock solution.
• Mixing: Gentle pipetting or inversion; avoid vortexing.
• Aliquoting: Single-use aliquots; avoid freeze-thaw.
• Storage: –20°C to –80°C for long-term; 2–8°C for short-term.

These steps will ensure optimal solubility, stability, and biological activity of recombinant rat Agrin for cell culture experiments.