Recombinant Human beta-Defensin 3

Recombinant Human beta-Defensin 3 (hBD-3) is a highly valuable tool for research due to its broad-spectrum biological activities and well-characterized roles in innate immunity. Here are the key reasons why you should consider using recombinant hBD-3 in your research applications:

1. Potent Antimicrobial Activity

• hBD-3 is a cationic host defense peptide with strong activity against a wide range of pathogens, including bacteria (Gram-positive and Gram-negative), fungi, and viruses.
• It disrupts microbial membranes, inhibits biofilm formation, and can act against antibiotic-resistant strains, making it relevant for studies on novel antimicrobial strategies.

2. Immunomodulatory Functions

• hBD-3 modulates immune responses by acting as a chemoattractant for immune cells (e.g., dendritic cells, T cells) and by regulating inflammatory pathways.
• It can suppress excessive inflammation by modulating Toll-like receptor signaling and cytokine production, which is useful for studies on immune regulation and inflammatory diseases.

3. Antiviral Properties

• hBD-3 has demonstrated antiviral activity against viruses such as HIV and herpes simplex virus.
• It can block viral entry and modulate host immune responses to infection, making it suitable for antiviral research and therapeutic development.

4. Wound Healing and Tissue Repair

• hBD-3 accelerates wound healing by promoting keratinocyte and fibroblast proliferation, migration, and activation.
• It enhances angiogenesis and tissue regeneration, supporting its use in studies on skin repair, chronic wounds, and regenerative medicine.

5. Stability and Reproducibility

• Recombinant hBD-3 is produced under controlled conditions, ensuring high purity, batch-to-batch consistency, and reliable experimental results.
• Its stability in biological fluids (such as serum) makes it suitable for both in vitro and in vivo studies.

6. Versatility in Research Models

• hBD-3 can be used in cell culture, animal models, and biochemical assays to study host-pathogen interactions, immune responses, wound healing, and antimicrobial mechanisms.
• It is also a useful scaffold for designing and testing novel peptide-based therapeutics.

7. Translational Potential

• Due to its multifunctional nature, hBD-3 is a promising candidate for developing new therapies against infectious diseases, chronic inflammation, and impaired wound healing.

In summary, recombinant Human beta-Defensin 3 is a versatile, well-characterized, and biologically active peptide that can significantly enhance the scope and impact of your research in immunology, microbiology, and regenerative medicine.

You can use recombinant human beta-Defensin 3 as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is of high purity, its concentration is accurately known, and it matches the sequence and structure recognized by the antibodies in your assay.

Supporting details and best practices:

• Recombinant proteins are commonly used as ELISA standards: Many commercial ELISA kits for human beta-defensin 3 (BD-3) use recombinant BD-3 as the calibrator or standard for generating the standard curve. This is standard practice in quantitative immunoassays.

• Purity and quantification: Ensure the recombinant BD-3 is highly purified and its concentration is accurately determined, typically by absorbance at 280 nm or amino acid analysis. Impurities or inaccurate quantification can affect the accuracy of your standard curve.

• Carrier protein considerations: Recombinant BD-3 is available in both carrier-free and carrier-added (e.g., BSA) forms. Carrier-free protein is preferred if you want to avoid potential interference from BSA or other additives, especially if your assay is sensitive to such components.

• Sequence and epitope compatibility: The recombinant BD-3 should have the same amino acid sequence (and, if relevant, post-translational modifications) as the native protein detected by your ELISA antibodies. Most commercial recombinant BD-3 proteins correspond to the mature peptide (typically Gly23-Lys67, 45 amino acids).

• Validation: If you are developing your own ELISA or using a kit not specifically validated with your recombinant standard, it is good practice to perform a recovery or parallelism test. This ensures that the recombinant standard behaves similarly to the endogenous protein in your sample matrix.

• Documentation: When reporting results, specify that recombinant human BD-3 was used as the standard, including its source, lot, and concentration determination method, to ensure reproducibility and transparency.

Summary Table: Use of Recombinant BD-3 as ELISA Standard

References:

• Recombinant BD-3 is routinely used as a calibrator in published ELISA protocols.
• Commercial ELISA kits specify the use of recombinant BD-3 as the standard.
• Carrier-free and carrier-added forms are available for different applications.

If you have a specific recombinant BD-3 preparation, verify its sequence, purity, and concentration before use. If your ELISA kit provides its own standard, use that for best compatibility unless you have validated your recombinant standard in the same system.

Recombinant Human beta-Defensin 3 (hBD-3) has been validated in published research for several key applications:

• Antimicrobial activity assays: hBD-3 has been extensively validated for its broad-spectrum antimicrobial effects against Gram-positive and Gram-negative bacteria, fungi, and some viruses, including multidrug-resistant strains such as MRSA and vancomycin-resistant Enterococcus faecium. These assays typically involve direct incubation of recombinant hBD-3 with microbial cultures to assess bactericidal or fungicidal activity.

• Immunomodulation and cell signaling studies: hBD-3 has been used to study its ability to modulate immune responses, including the activation of monocytes, mast cells, and dendritic cells, as well as the induction of cytokine production (e.g., IL-6, IFN-γ, IL-12p70, IL-10, IFN-α). It has also been shown to promote chemotaxis of immune cells and enhance cellular uptake of nucleic acids, which is relevant for vaccine adjuvant research.

• Wound healing and tissue regeneration models: Recombinant hBD-3 has been validated for its role in stimulating angiogenesis, extracellular matrix production, and keratinocyte migration, supporting its application in wound healing and tissue regeneration studies.

• Antiviral assays: hBD-3 has demonstrated inhibitory effects on viral infectivity, including direct interactions with HIV-1 and HSV, and protective effects against influenza virus by coating target cells.

• Bioassays for cell signaling pathways: hBD-3 has been used to study its impact on intracellular signaling, such as the stabilization and activation of β-catenin in epithelial cells during viral infection (e.g., RSV). These studies often involve western blotting, reporter assays, and siRNA-mediated gene silencing.

• Vaccine adjuvant research: hBD-3 has been validated as an adjuvant to enhance antigen-specific immune responses by promoting the uptake of nucleic acids and boosting both cellular and humoral immunity in animal models.

• Nanomedicine and drug delivery: Its unique physicochemical properties have been explored for targeted therapy and improved drug delivery systems, although these applications are more recent and may be less widely validated.

• Transgenic animal models: Recombinant hBD-3 has been expressed in transgenic animals (e.g., goats) to assess its antibacterial activity in milk and other tissues.

Common experimental protocols include:

• Microbial killing assays (CFU reduction, MIC determination)
• Cytokine quantification (ELISA, multiplex assays)
• Chemotaxis and cell migration assays
• Western blotting and immunofluorescence for signaling studies
• In vivo infection and wound healing models
• Vaccine efficacy studies in animal models

Summary Table:

These applications are supported by multiple peer-reviewed studies, demonstrating the versatility of recombinant hBD-3 in both basic and translational research contexts.

To reconstitute and prepare Recombinant Human beta-Defensin 3 (hBD-3) protein for cell culture experiments, follow these best-practice steps:

1. Centrifuge the vial before opening to ensure all lyophilized protein is at the bottom.

2. Reconstitution solvent:

   • The most widely recommended solvent is sterile distilled water or sterile 10 mM acetic acid.
   • If using water, you may add 0.1% BSA as a carrier to stabilize the protein, especially for low concentrations or long-term storage.
   • Some formulations may require reconstitution in 4 mM HCl or other specific buffers; always check the product datasheet for formulation-specific instructions.

3. Concentration:

   • Reconstitute to a final concentration of 0.1–1.0 mg/mL.
   • For example, add 100–1000 μL of solvent per 0.1 mg of protein to achieve this range.

4. Dissolving the protein:

   • Gently pipette up and down and wash down the sides of the vial to ensure full recovery.
   • Do not vortex or shake vigorously, as this can denature the protein.

5. Aliquoting and storage:

   • After reconstitution, aliquot the solution to avoid repeated freeze-thaw cycles.
   • Store aliquots at 2–8°C for up to 1 week or at –20°C to –80°C for up to 3 months.

6. Dilution for cell culture:

   • Before adding to cells, dilute the reconstituted stock into your cell culture medium to the desired working concentration.
   • If the stock was reconstituted in acetic acid or HCl, ensure the final acid concentration in the culture medium is negligible and does not affect cell viability. A typical approach is to dilute at least 1:1000 into medium, or perform a buffer exchange if needed.

7. General precautions:

   • Use sterile technique throughout to avoid contamination.
   • Wear appropriate PPE (lab coat, gloves) when handling recombinant proteins.

Summary Table: Key Steps for hBD-3 Reconstitution

Note: Always consult the specific product datasheet for any unique formulation or reconstitution requirements, as excipients or buffer components may vary between suppliers.

If you need a protocol for a specific downstream application (e.g., antimicrobial assay, cytokine induction), please specify for tailored guidance.