Recombinant Human Beta Defensin-2 (BD-2)

Recombinant Human Beta Defensin-2 (BD-2) is widely used in research due to its potent antimicrobial, immunomodulatory, and tissue-protective properties, making it valuable for studies in host defense, inflammation, wound healing, and therapeutic development.

Key reasons to use recombinant BD-2 in research applications:

• Antimicrobial Activity: BD-2 exhibits broad-spectrum activity against bacteria, fungi, and some viruses, including drug-resistant strains. This makes it a useful tool for studying innate immune responses and developing novel antimicrobial strategies.

• Immunomodulation: BD-2 can modulate immune responses by recruiting and activating immune cells such as dendritic cells, monocytes, and T-cells, primarily through interactions with chemokine receptors CCR2 and CCR6. It suppresses proinflammatory cytokine secretion (e.g., TNF-α, IL-12, IL-1β) and can attenuate excessive inflammation, which is relevant for research in inflammatory diseases and immune regulation.

• Wound Healing and Tissue Regeneration: BD-2 promotes wound healing and angiogenesis, both in vitro and in vivo, by modulating inflammatory pathways and supporting tissue repair processes. It has been shown to accelerate healing in models of chronic wounds, such as diabetic ulcers, by inhibiting detrimental enzymes like MMP-9 and PKC-βII.

• Barrier Protection: BD-2 protects epithelial barriers by modulating protease activity and supporting the integrity of tissues such as skin and mucosa, which is important for studies on barrier function and epithelial biology.

• Low Cytotoxicity: BD-2 demonstrates minimal toxicity toward mammalian cells, making it suitable for in vitro and in vivo studies without significant off-target effects.

• Therapeutic Potential: Recombinant BD-2 is being explored as a candidate for immunotherapy, anti-infective therapy, and as an adjunct in cancer research due to its ability to enhance anti-tumor immune responses and modulate the tumor microenvironment.

• Experimental Versatility: Recombinant BD-2 is suitable for a range of applications, including cell culture, animal models, and biochemical assays, allowing for mechanistic studies and therapeutic evaluation.

In summary, recombinant BD-2 is a versatile research tool for investigating innate immunity, antimicrobial defense, inflammation, tissue repair, and therapeutic development, with broad applications in immunology, microbiology, and regenerative medicine.

Yes, recombinant human Beta Defensin-2 (BD-2) can be used as a standard for quantification or calibration in ELISA assays, provided it is compatible with your assay antibodies and protocol.

Key considerations and supporting details:

• Intended Use: Recombinant human BD-2 is commonly used as a standard in ELISA applications. For example, it is specifically recommended as a standard for ELISA when paired with appropriate anti-BD-2 antibodies.
• Purity and Activity: High-purity recombinant BD-2 (>98% by SDS-PAGE and HPLC) is suitable for quantitative applications, and its activity is typically validated by functional assays.
• Reconstitution and Handling: Follow the manufacturer’s instructions for reconstitution (e.g., with 10 mM acetic acid) and storage to maintain protein integrity.
• Antibody Compatibility: Ensure that the antibodies used in your ELISA recognize both recombinant and native forms of BD-2. Most commercial ELISA kits use standards that are either recombinant or synthetic BD-2, and their antibodies are validated for these forms.
• Standard Curve Preparation: Prepare a dilution series of the recombinant BD-2 in the same buffer as your samples to generate a standard curve for quantification.
• Research Use Only: Recombinant BD-2 standards are for research use and not for diagnostic purposes.

Additional notes:

• Some ELISA kits specify that their standards are recombinant BD-2, while others may use native protein. Always check your kit’s documentation to ensure compatibility.
• If your ELISA kit is designed for native BD-2 only, confirm with the kit provider or by reviewing the kit manual whether recombinant BD-2 is suitable as a standard.

In summary, recombinant human BD-2 is widely used and accepted as a standard for ELISA quantification, as long as it is compatible with your assay system and antibodies.

Recombinant Human Beta Defensin-2 (BD-2) has been validated in published research for a range of applications, primarily in the fields of immunology, infectious disease, wound healing, and epithelial barrier protection.

Key validated applications include:

• Antimicrobial activity assays: BD-2 is widely used to assess its ability to disrupt microbial membranes and inhibit the growth of bacteria and fungi, reflecting its role in innate immunity.
• Immunomodulation studies: Recombinant BD-2 has been validated for its capacity to modulate immune responses, including suppression of pro-inflammatory cytokine production (e.g., TNF-α, IL-6, IL-1β) in human peripheral blood mononuclear cells (PBMCs) and mouse macrophages.
• In vivo models of inflammatory disease: Systemic administration of recombinant BD-2 has been shown to ameliorate inflammation in multiple animal models of inflammatory bowel disease (IBD), including DSS-induced, TNBS-induced, and T-cell transfer colitis models.
• Wound healing and tissue regeneration: BD-2 has been validated for promoting wound closure and tissue regeneration, particularly in the context of diabetic wounds and bone defect repair, where it enhances osteogenic marker expression and supports angiogenesis.
• Epithelial barrier protection: BD-2 has been used in vitro to protect keratinocyte barrier function against protease-mediated damage, relevant to skin diseases such as atopic dermatitis.
• Chemotaxis and cell signaling: BD-2 acts as a chemoattractant for dendritic cells, monocytes, and T-cells via chemokine receptors CCR2 and CCR6, and modulates signaling pathways such as NF-κB and CREB phosphorylation.
• Lung inflammation models: Recombinant BD-2 has been validated in studies showing attenuation of lung neutrophilia and preservation of immune competence in cigarette smoke-induced lung injury models.

Experimental techniques and protocols where recombinant BD-2 has been validated include:

• Functional antimicrobial assays (bacterial/fungal killing)
• Cytokine quantification (ELISA, qPCR)
• Cell culture assays (e.g., PBMCs, keratinocytes, bone marrow stromal cells)
• Animal models (systemic and local administration in mice)
• Barrier function assays (e.g., transepithelial electrical resistance, protease challenge)
• Histological and molecular analysis of tissue regeneration and inflammation

These applications are supported by both in vitro and in vivo studies, with recombinant BD-2 serving as a tool to dissect its biological functions and therapeutic potential in host defense, immune regulation, and tissue repair.

To reconstitute and prepare Recombinant Human Beta Defensin-2 (BD-2) protein for cell culture experiments, follow these general guidelines based on manufacturer recommendations and best practices:

Reconstitution

1. Centrifuge the vial briefly before opening to ensure all lyophilized powder is at the bottom.
2. Reconstitution Solution:
   • Most manufacturers recommend reconstituting with sterile, distilled water (18 MΩ-cm H₂O) or 10 mM acetic acid.
   • Some suggest using 0.1% BSA in aqueous buffer to stabilize the protein, especially for extended storage.
   • Bio-Rad and Reprokine specifically recommend 10 mM acetic acid.
   • Prospec Bio and Abcam recommend sterile water or 0.1% BSA aqueous buffer.
3. Concentration:
   • Reconstitute to a concentration of not less than 100 µg/mL (0.1 mg/mL).
   • For example, add 100 µL of solvent to 10 µg of protein to achieve 100 µg/mL.
4. Mixing:
   • Gently mix or swirl the vial after reconstitution. Avoid vigorous shaking to prevent denaturation.

Preparation for Cell Culture

1. Further Dilution:
   • Dilute the reconstituted stock in cell culture medium or appropriate buffer to the desired working concentration (typically 10–100 ng/mL for functional assays).
2. Sterility:
   • Ensure all solutions and handling are performed under sterile conditions to avoid contamination.

Storage

1. Short-term:
   • Reconstituted protein can be stored at 4°C for up to one week.
2. Long-term:
   • Aliquot into small working volumes and store at –20°C (avoid frost-free freezers).
   • Avoid repeated freeze-thaw cycles.
3. Additives:
   • For extended storage, consider adding 0.1% BSA to prevent adsorption to vial walls.

Additional Tips

• Centrifuge the reconstituted solution before use if a precipitate is observed.
• Always refer to the lot-specific datasheet for any product-specific instructions.

Summary Protocol

1. Centrifuge vial.
2. Reconstitute with 10 mM acetic acid or sterile water to ≥100 µg/mL.
3. Gently mix.
4. Dilute in culture medium for experiments.
5. Store aliquots at –20°C for long-term use.

These steps will help ensure the stability and activity of Recombinant Human Beta Defensin-2 for your cell culture experiments.