Recombinant Rat MIP-1α

Recombinant rat MIP-1α (also designated CCL3) is a valuable tool for inflammatory and immunological research due to its well-characterized biological functions and versatile applications.

Biological Activity and Mechanism

Recombinant rat MIP-1α functions as a potent chemotactic chemokine that activates specific cell populations through binding to chemokine receptors CCR1, CCR4, and CCR5. This receptor engagement triggers inflammatory responses and recruitment of immune cells. The protein exhibits dose-dependent chemotactic activity for both monocytes and neutrophils, with maximal in vitro activity observed at approximately 50 nM concentration.

Research Applications

Inflammatory and Pulmonary Models

The recombinant protein is particularly useful for studying acute lung injury and inflammatory pathways. In vivo studies demonstrate that intratracheal instillation of recombinant rat MIP-1α results in significant cell influx into lung airspace, primarily recruiting monocytes and macrophages. This makes it valuable for investigating neutrophil-dependent inflammatory models and understanding the role of MIP-1α in acute pulmonary inflammation.

Immune Cell Recruitment Studies

Beyond pulmonary applications, recombinant rat MIP-1α enables investigation of leukocyte migration and inflammatory cell recruitment mechanisms. The protein's ability to recruit granulocytes and induce neutrophil superoxide production makes it suitable for studying innate immune responses and cell-cell interactions during inflammation.

Wound Healing and Tissue Repair

MIP-1α may promote wound healing activity by recruiting macrophages to sites of tissue injury, making the recombinant form useful for investigating tissue repair mechanisms and macrophage-dependent healing processes.

Hematopoietic Regulation

Beyond its proinflammatory activities, recombinant rat MIP-1α inhibits hematopoietic stem cell proliferation, providing a tool for studying stem cell regulation and hematopoietic differentiation pathways.

Technical Advantages

The recombinant protein is produced as a non-glycosylated form containing 69 amino acids with a molecular mass of 7.8 kDa, ensuring consistency and reproducibility across experiments. The protein demonstrates stability when stored appropriately with carrier proteins at -20°C, facilitating long-term experimental planning and batch consistency.

Yes, recombinant Rat MIP-1α can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately determined. Recombinant proteins are commonly used as standards in quantitative ELISA protocols for cytokines and chemokines, including MIP-1α (CCL3).

Supporting details:

• ELISA kits for Rat MIP-1α are designed to detect both natural and recombinant forms of the protein, and their standard curves are typically generated using recombinant MIP-1α.
• Guidelines for ELISA standards recommend using a purified recombinant protein to prepare the standard curve, ensuring accurate quantification. The concentration of the recombinant standard should be verified, ideally by an orthogonal method such as HPLC or UV absorbance.
• Recombinant Rat MIP-1α is available in formulations suitable for use as ELISA standards, sometimes with carrier proteins like BSA to enhance stability. The protein should be reconstituted and diluted according to the ELISA kit instructions to match the assay’s dynamic range.
• Validation and specificity: Most commercial ELISA kits validate their quantification using recombinant standards, and the assay specificity includes both natural and recombinant forms.

Best practices:

• Confirm the recombinant protein’s purity and identity (e.g., by SDS-PAGE and mass spectrometry).
• Accurately determine the protein concentration before preparing serial dilutions for the standard curve.
• Follow the ELISA kit’s protocol for standard preparation, including recommended buffers and diluents.
• Include internal controls and matrix-matched standards if possible, to account for potential matrix effects.

In summary: Recombinant Rat MIP-1α is suitable for use as a standard in ELISA quantification, provided it is properly characterized and prepared according to best practices for quantitative immunoassays.

Recombinant Rat MIP-1α (CCL3) has been validated for several key applications in published research, including functional assays, ELISA, Western blot, and immunohistochemistry.

Validated Applications and Published Uses:

• Functional Assays:
  Recombinant rat MIP-1α has been used to demonstrate dose-dependent chemotactic activity for rat and human monocytes and neutrophils, confirming its biological activity in vitro and in vivo. It has also been used to study its role in immune cell recruitment and inflammatory responses.

• ELISA (Enzyme-Linked Immunosorbent Assay):
  The protein is commonly used as a standard or control in ELISA to quantify MIP-1α levels in biological samples, such as bronchoalveolar lavage fluid or plasma, particularly in models of inflammation, infection, or tissue injury.

• Western Blot:
  Detection and quantification of MIP-1α protein in tissue extracts or biological fluids have been performed using Western blot, with recombinant rat MIP-1α serving as a positive control or standard.

• Immunohistochemistry:
  Recombinant rat MIP-1α has been used to generate or validate antibodies for immunohistochemical detection of MIP-1α in tissue sections, enabling localization studies in rat tissues.

Additional Research Applications:

• Disease Models:
  Recombinant rat MIP-1α has been used to investigate its role in acute lung injury, where it was shown to mediate neutrophil recruitment and contribute to tissue damage in rat models of immune complex- and LPS-induced lung injury.
  It has also been implicated in studies of bone resorption, periodontitis, and as a biomarker in various inflammatory and infectious diseases.

• Bioactivity Assays:
  The protein is used in cell-based assays to assess chemotaxis, cytokine production, and signaling pathway activation in immune cells.

• Multiplex Cytokine Profiling:
  MIP-1α is included in multiplex bead-based immunoassays (e.g., Luminex) for profiling cytokine responses in disease models, including malaria and sepsis.

Summary Table:

These applications are supported by both product validation data and peer-reviewed research, confirming the utility of recombinant rat MIP-1α in immunological, inflammatory, and disease model studies.

To reconstitute and prepare Recombinant Rat MIP-1α (CCL3) protein for cell culture experiments, dissolve the lyophilized protein in sterile, high-purity water (18 MΩ-cm H₂O) at a concentration of at least 100 µg/mL. After reconstitution, further dilute the protein in appropriate cell culture media or buffer for your experimental needs.

Step-by-step protocol:

• Preparation:

  • Briefly centrifuge the vial to collect the lyophilized powder at the bottom.
  • Use sterile technique throughout to avoid contamination.

• Reconstitution:

  • Add sterile 18 MΩ-cm H₂O to achieve a concentration of ≥100 µg/mL (e.g., add 100 µL water to 100 µg protein).
  • Gently mix by pipetting or swirling; avoid vigorous vortexing to prevent protein denaturation.
  • If the protein does not fully dissolve, allow it to sit at room temperature for a few minutes, then gently mix again.

• Dilution:

  • Once fully dissolved, dilute the stock solution into your desired buffer or cell culture medium. Commonly used buffers include PBS or cell culture media supplemented with serum or carrier protein (e.g., 0.1% BSA or HSA) to stabilize the protein and prevent adsorption to plasticware.
  • Prepare working concentrations according to your experimental design (e.g., 10–100 ng/mL for chemotaxis assays).

• Storage:

  • Store the reconstituted protein at 4°C for up to one week.
  • For long-term storage, aliquot and freeze at –20°C or below. Avoid repeated freeze-thaw cycles by aliquoting into single-use volumes.
  • For extended stability, add a carrier protein (e.g., 0.1% BSA or HSA) before freezing.

Additional notes:

• The protein is typically supplied as a sterile, lyophilized powder with no additives.
• Do not use the protein for applications outside laboratory research.
• Confirm biological activity with a functional assay (e.g., chemotaxis of rat macrophages at 60 ng/mL).

Summary Table:

Always consult the specific product datasheet for any additional recommendations or requirements.