Recombinant Human CCL4L1

Recombinant Human CCL4L1 is used in research applications to study chemokine-mediated immune cell recruitment, HIV entry inhibition, and inflammation signaling, due to its specific interaction with the CCR5 receptor and its close homology to CCL4 (MIP-1β).

Key scientific reasons to use Recombinant Human CCL4L1:

• Chemotaxis Assays: CCL4L1 is a potent chemoattractant for lymphocytes, NK cells, monocytes, and immature dendritic cells, making it valuable for studying immune cell migration and inflammatory responses.
• CCR5 Receptor Studies: CCL4L1 binds CCR5, a receptor critical for immune cell trafficking and a co-receptor for HIV entry. CCL4L1 can be used to investigate CCR5-mediated signaling and to model HIV suppression mechanisms, as it blocks HIV entry into CCR5-expressing cells.
• Comparative Isoform Analysis: CCL4L1 is a paralog of CCL4, sharing >98% amino acid identity, but with distinct genetic origins and possible functional nuances. Using recombinant CCL4L1 allows for direct comparison of isoform-specific effects in cellular assays.
• Bioassays and Functional Validation: Recombinant CCL4L1 is validated for bioactivity, ensuring reliable results in cell-based assays, chemotaxis experiments, and cytokine signaling studies.
• Human-Specific Research: Recombinant proteins produced in E. coli or human cell lines provide high purity and consistency, enabling reproducible results in human cell models.

Typical applications include:

• Immune cell migration and chemotaxis assays
• HIV research, especially CCR5-mediated viral entry inhibition
• Inflammatory signaling pathway studies
• ELISA standards and positive controls for chemokine detection
• Comparative studies of chemokine isoforms and their biological effects

Technical considerations:

• Recombinant CCL4L1 is available in carrier-free and carrier-added formulations, allowing flexibility for cell culture, ELISA, or biochemical assays depending on experimental needs.
• The protein is typically supplied as a lyophilized powder, with recommended reconstitution protocols to maintain activity and stability.

Using recombinant human CCL4L1 enables precise, reproducible investigation of chemokine biology, immune cell trafficking, and HIV-related mechanisms in human-relevant systems, supporting both basic and translational research objectives.

Recombinant Human CCL4L1 can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated for parallelism and specificity with your assay system. Recombinant proteins are commonly employed as standards in ELISA protocols to generate calibration curves for quantifying target analytes in biological samples.

Key considerations for use:

• Validation of Parallelism: It is essential to confirm that the recombinant CCL4L1 standard produces a dose-response curve parallel to that of endogenous CCL4L1 in your sample matrix. This ensures accurate quantification and comparability between standard and sample signals.
• Purity and Formulation: The recombinant CCL4L1 should be of high purity (typically >95% by SDS-PAGE) and formulated appropriately (e.g., with or without carrier proteins such as BSA) to match your assay requirements.
• Epitope Recognition: The antibodies used in your ELISA must recognize both the recombinant and native forms of CCL4L1. Sequence identity between recombinant and endogenous proteins is typically high (>98%), but confirm that the recombinant construct includes all relevant epitopes for antibody binding.
• Standard Curve Preparation: Prepare serial dilutions of the recombinant CCL4L1 to generate a standard curve covering the expected concentration range in your samples. Use appropriate curve fitting (e.g., four- or five-parameter logistic regression) for quantification.

Best Practices:

• Run the recombinant standard in parallel with biological samples to assess curve parallelism and matrix effects.
• Validate the assay for accuracy, precision, and specificity using the recombinant standard before routine quantification.
• Store and handle the recombinant protein according to manufacturer recommendations to maintain stability and activity.

Summary:
Recombinant Human CCL4L1 is suitable as an ELISA standard if validated for your specific assay. Ensure parallelism, purity, and antibody compatibility for reliable quantification.

Recombinant Human CCL4L1 has been validated in published research primarily for applications involving bioassays of chemotactic activity, functional assays, and as a positive control in immunological studies. Its main uses are based on its biological activity as a chemokine, particularly its ability to induce chemotaxis in CCR5- or CCR1-expressing cells and to inhibit HIV replication in CCR5+ monocytes.

Key validated applications include:

• Bioassays/Functional Assays:
  Used to assess chemotactic responses of immune cells (such as monocytes, lymphocytes, NK cells, and dendritic cells) via CCR5 or CCR1 signaling. These assays typically measure cell migration in response to CCL4L1 gradients.

• HIV Inhibition Studies:
  Employed to study its role as an HIV-suppressive factor, specifically its ability to block HIV entry into CCR5-expressing cells.

• Positive Control in Immunological Assays:
  Used as a positive control for chemotaxis and functional studies in immunology, including as a standard in ELISA and Western blotting, and for validating cell migration protocols.

• Cell Culture Applications:
  Utilized in cell culture systems to study chemokine-driven signaling, immune cell recruitment, and inflammatory responses.

• Blocking/Neutralization Assays:
  Applied in blocking assays to investigate the inhibition of chemokine activity or receptor binding, often in the context of immune modulation or drug screening.

• SDS-PAGE and Western Blot:
  Used as a molecular standard or positive control for protein detection and quantification in SDS-PAGE and Western blot analyses.

Summary Table: Validated Applications for Recombinant Human CCL4L1

Additional Notes:

• The protein is often used in its carrier-free or BSA-formulated forms depending on the application (e.g., cell culture vs. ELISA standard).
• Published research and product datasheets consistently validate its use in chemotaxis and HIV inhibition assays, reflecting its biological relevance as a chemokine.

If you require details on a specific application protocol or a particular cell type, please specify for more targeted information.

To reconstitute and prepare Recombinant Human CCL4L1 protein for cell culture experiments, dissolve the lyophilized protein at 100 μg/mL in sterile PBS; if the protein is supplied with a carrier, ensure the PBS contains at least 0.1% human or bovine serum albumin (BSA) to enhance stability. For carrier-free formulations, reconstitute directly in sterile PBS without BSA.

Detailed protocol and best practices:

• Preparation:

  • Before opening the vial, briefly centrifuge it to collect all lyophilized material at the bottom.
  • Warm the vial to room temperature before opening to minimize condensation.

• Reconstitution:

  • For carrier-containing formulations: Add sterile PBS with at least 0.1% BSA to achieve a final concentration of 100 μg/mL.
  • For carrier-free formulations: Add sterile PBS only, to the same concentration.
  • Alternatively, some protocols allow reconstitution in sterile water, but PBS with BSA is preferred for cell culture to maintain protein stability and activity.
  • Gently pipette to mix; do not vortex or shake vigorously, as this may denature the protein.
  • Allow the solution to sit at room temperature for 20–30 minutes to ensure complete dissolution.

• Aliquoting and Storage:

  • Once fully dissolved, aliquot the solution to avoid repeated freeze-thaw cycles, which can degrade the protein.
  • Store aliquots at –20°C to –70°C for long-term storage; short-term storage (up to 1 month) at 2–8°C is acceptable.
  • Avoid storing working solutions at concentrations below 0.1 mg/mL for extended periods; use immediately or add carrier protein if further dilution is needed.

• Working Solution Preparation:

  • Dilute the stock solution to the desired working concentration using cell culture medium or buffer containing 0.1% BSA, 5% HSA, or 10% FBS to prevent adsorption and maintain activity.
  • Do not dilute with pure water, as this may cause protein degradation.

• Quality Control:

  • Confirm protein solubility visually; the solution should be clear.
  • Optionally, verify protein integrity by SDS-PAGE if required.

Key technical notes:

• Do not vortex or use vigorous mixing at any step.
• Avoid repeated freeze-thaw cycles by aliquoting.
• Use sterile technique throughout to prevent contamination.
• For cell-based assays, ensure endotoxin levels are appropriate for your application (typically <1 EU/μg protein).

This protocol ensures optimal solubility, stability, and biological activity of recombinant CCL4L1 for cell culture experiments.