Recombinant Human NAP-2 (CXCL7)

Recombinant Human NAP-2 (CXCL7) is widely used in research applications due to its critical role as a chemokine that regulates immune cell migration, inflammation, and tissue repair.

Key scientific reasons to use recombinant NAP-2 (CXCL7):

• Neutrophil Activation and Chemotaxis: NAP-2 binds to the CXCR2 receptor, inducing potent chemoattraction and activation of neutrophils, making it essential for studies on innate immunity, inflammation, and leukocyte trafficking.
• Functional Assays: It is used to assess neutrophil functions such as chemotaxis, degranulation, and myeloperoxidase release in vitro, providing a controlled system to study cellular responses to chemokines.
• MSC Migration: NAP-2 stimulates migration of mesenchymal stem cells (MSCs) via CXCR2, relevant for research on tissue regeneration and stem cell homing.
• Angiogenesis and Wound Healing: As an angiogenic chemokine, NAP-2 is involved in endothelial cell recruitment and vascular repair, making it valuable for studies on tissue remodeling and healing.
• Inflammatory Disease Models: Elevated NAP-2 levels are associated with acute coronary syndromes and other inflammatory conditions, supporting its use in cardiovascular and immunological disease models.
• Antibacterial Activity: NAP-2 exhibits direct antibacterial effects against pathogens such as Streptococcus pyogenes, useful for host-pathogen interaction studies.
• Megakaryocytopoiesis Regulation: NAP-2 inhibits megakaryocyte formation, relevant for hematopoiesis and platelet biology research.

Typical research applications include:

• Chemotaxis assays (e.g., Boyden chamber, transwell migration)
• Neutrophil activation and degranulation studies
• ELISA and immunoassay development for biomarker quantification
• Functional assays in cell culture and whole blood
• Disease modeling (inflammation, cardiovascular, wound healing)
• Stem cell migration and tissue engineering studies

Advantages of recombinant protein:

• High purity and reproducibility for quantitative and mechanistic studies
• Defined activity, enabling dose-response and mechanistic experiments
• Absence of confounding factors present in native preparations

Using recombinant human NAP-2 (CXCL7) allows precise investigation of chemokine-mediated cellular processes, immune responses, and disease mechanisms in a controlled experimental setting.

Yes, recombinant human NAP-2 (CXCL7) can be used as a standard for quantification and calibration in ELISA assays. This is a well-established practice in immunoassay development.

Suitability as an ELISA Standard

Recombinant NAP-2/CXCL7 proteins are specifically designed and utilized as calibration standards in sandwich ELISA systems. The recombinant protein is a 7.6 kDa molecule consisting of 70 amino acids that maintains the biological activity and immunological properties necessary for accurate quantification. Many commercial ELISA development kits include recombinant NAP-2/CXCL7 as a standard component, confirming its validity for this application.

Standard Curve Development

When preparing your standard curve, the recombinant protein should be diluted in an appropriate diluent—typically 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at pH 7.2-7.4. The standard curve range varies depending on your specific assay configuration, but typical detection ranges span from approximately 0.78 ng/mL to 50 ng/mL, with some assays achieving sensitivities as low as 0.51 pg/mL.

Key Considerations

Sample matrix compatibility: The diluent used for your standards should match the matrix of your samples. While the suggested diluent works well for cell culture supernatant samples, complex matrices such as serum and plasma may require evaluation and optimization of the diluent prior to use.

Assay precision: When properly prepared and used according to established protocols, recombinant NAP-2/CXCL7 standards typically yield inter-assay coefficients of variation below 12% and intra-assay coefficients of variation below 10%, demonstrating reliable quantification.

Cross-reactivity: Ensure that your detection antibodies demonstrate minimal cross-reactivity with other CXC chemokines (such as CXCL1, CXCL2, CXCL3, CXCL5, and CXCL6) to maintain assay specificity.

Recombinant Human NAP-2 (CXCL7) has been validated for a range of applications in published research, primarily related to its role as a chemokine in inflammation, neutrophil recruitment, and as a biomarker in disease contexts.

Validated Applications in Published Research:

• Chemotaxis and Functional Assays:
  Recombinant NAP-2 (CXCL7) is widely used to study neutrophil chemotaxis and activation, including assays measuring directed migration, degranulation, and myeloperoxidase release from neutrophils. It is also used to assess activation of other cell types such as monocytes and endothelial progenitor cells.

• Receptor Binding and Signal Transduction:
  CXCL7 is validated for studies involving CXCR2 receptor binding and downstream signaling, including activation of pathways such as NF-κB in various cell types.

• Blocking/Neutralization Studies:
  Recombinant NAP-2 is used in blocking and neutralization assays to evaluate the specificity and function of anti-CXCL7 antibodies and to dissect its biological activity in vitro.

• ELISA and Immunoassays:
  It is a standard for ELISA and other immunoassays to quantify CXCL7 levels in biological samples (e.g., plasma, serum) for both basic research and biomarker studies.

• Western Blot:
  Used as a positive control or standard in Western blotting to detect CXCL7 in cell lysates or tissue extracts.

• Immunohistochemistry (IHC):
  Recombinant CXCL7 is used for IHC and IHC-Paraffin protocols, often as a control or for antibody validation.

• RNAi Validation:
  It has been used in RNA interference (RNAi) validation experiments to confirm knockdown specificity.

• Proteomics and Biomarker Discovery:
  CXCL7/NAP-2 has been identified and validated as a biomarker in proteomic studies for early detection of cancers such as lung cancer and colorectal cancer, using immunoassays and mass spectrometry.

• Antibacterial Activity Assays:
  Recombinant NAP-2 has been used in bactericidal assays to compare its direct antibacterial activity against pathogens such as Streptococcus pyogenes.

Key Research Contexts:

• Inflammation and Wound Healing:
  NAP-2 is used to model and dissect mechanisms of inflammatory cell recruitment and wound healing in vitro and in vivo.

• Cancer Research:
  It is validated as a diagnostic and prognostic biomarker in cancer, particularly for early detection of lung and colorectal cancers.

• Platelet Biology:
  Used to study platelet-derived chemokine release and its effects on neutrophil-platelet interactions and thrombus formation.

Summary Table of Applications

These applications are supported by both primary research articles and product validation data, reflecting the broad utility of recombinant human NAP-2 (CXCL7) in immunology, cell biology, and translational research.

Reconstitution Guidelines

Recombinant Human NAP-2 (CXCL7) is typically supplied as a lyophilized powder and requires proper reconstitution before use in cell culture experiments. The reconstitution concentration and buffer composition depend on the specific formulation and intended application.

Standard Reconstitution Procedures

Recommended Reconstitution Concentrations:

The protein should be reconstituted to a concentration between 0.1–1.0 mg/mL in sterile distilled water or an aqueous buffer containing 0.1% bovine serum albumin (BSA). Some formulations recommend reconstituting at 100 μg/mL in sterile phosphate-buffered saline (PBS), while others specify 0.1 mg/mL in sterile water.

Important Reconstitution Steps:

Before opening the vial, centrifuge the product for 20–30 seconds using a tabletop centrifuge to collect any powder that may have adhered to the cap or walls. This ensures you recover the full amount of protein. When dissolving the lyophilized powder, do not vortex the solution, as vigorous mixing can cause protein aggregation and denaturation. Instead, gently mix by pipetting or gentle swirling until the powder is completely dissolved.

Storage and Stability

Post-Reconstitution Storage

After reconstitution under aseptic conditions, the protein exhibits different stability profiles depending on temperature:

• Short-term storage (2–8°C): 1 month under sterile conditions
• Long-term storage (−20 to −70°C): 3–6 months under sterile conditions
• Lyophilized powder (−20°C): At least 2 years from date of receipt

Critical Storage Practices

Use a manual defrost freezer and avoid repeated freeze-thaw cycles, as these can significantly reduce protein activity and stability. To minimize freeze-thaw damage, prepare small working aliquots of the reconstituted protein and freeze them separately for individual experiments. This approach allows you to thaw only the amount needed for each use without repeatedly exposing the entire stock to temperature fluctuations.

Preparation for Cell Culture Applications

Buffer Composition Considerations

For cell culture experiments, the reconstituted protein can be diluted into the specific buffer or cell culture medium required for your experimental protocol. If using a formulation that includes BSA as a carrier protein, this provides additional stability during storage and handling. Formulations without carrier protein may require the addition of BSA (0.1%) to the reconstitution buffer to maintain protein stability.

Quality Control

Ensure that the reconstituted solution is completely dissolved and homogeneous before use. The protein should appear as a clear solution; any visible particulates or cloudiness may indicate aggregation and should be avoided in experiments. For applications requiring precise protein concentrations, such as dose-response studies or chemotaxis assays, prepare a standard detection curve for each experiment to account for any variations in protein activity.

The recombinant protein is suitable for mammalian cell culture applications and typically exhibits endotoxin levels below 0.1 EU/μg, making it appropriate for sensitive cell-based assays.