Recombinant Human TIMP-1

Recombinant Human TIMP-1 is widely used in research applications due to its well-characterized ability to inhibit matrix metalloproteinases (MMPs), regulate tissue remodeling, and modulate cellular processes such as proliferation, apoptosis, and immune responses.

Key scientific reasons to use recombinant TIMP-1 include:

• Specific and Potent MMP Inhibition: TIMP-1 is a natural inhibitor of several MMPs, including MMP-1, MMP-2, and MMP-9, with nanomolar IC50 values, making it essential for studies on extracellular matrix turnover, tissue remodeling, and related pathologies such as cancer, fibrosis, and arthritis.
• Defined Bioactivity and Purity: Recombinant TIMP-1 is produced with high purity and low endotoxin levels, ensuring reproducibility and minimizing confounding effects in cell-based and in vivo assays.
• Versatility in Experimental Applications: It is suitable for a range of applications, including:
  • Enzyme inhibition assays to study MMP activity.
  • Cell culture and tissue models to investigate effects on cell proliferation, apoptosis, and differentiation.
  • Bioassays and immunoassays as a standard or control.
  • Functional studies on immune cell activation, such as monocyte activation via interaction with amyloid precursor protein (APP), which has implications for inflammation and cancer research.
• Role in Disease Models: TIMP-1 is implicated in wound healing, tumor metastasis, angiogenesis, and inflammatory responses, making it valuable for mechanistic studies and therapeutic target validation in these areas.
• Cytokine-like Functions: Beyond MMP inhibition, TIMP-1 can trigger signaling pathways affecting immune responses and cell survival, broadening its utility in immunology and cell biology research.

Best practices for using recombinant TIMP-1:

• Reconstitute in sterile water at recommended concentrations for optimal activity.
• Select carrier-free preparations for sensitive assays or those requiring minimal additives.
• Confirm bioactivity using standardized inhibition assays before experimental use.

In summary, recombinant human TIMP-1 provides a reliable, bioactive tool for dissecting MMP-dependent and independent pathways in diverse biological and disease contexts, supporting mechanistic, translational, and therapeutic research.

Yes, recombinant human TIMP-1 can be used as a standard for quantification or calibration in ELISA assays, provided it is properly formulated and validated for this purpose. Recombinant TIMP-1 is commonly used as a standard in commercial human TIMP-1 ELISA kits, where it enables the generation of standard curves for quantitative measurement of TIMP-1 in biological samples.

Key considerations for use:

• Source and Purity: The recombinant TIMP-1 standard should be of high purity (typically >95%) and produced in a system that ensures correct folding and post-translational modifications, such as HEK293 or NS0 cells.
• Formulation: Standards are often supplied lyophilized and should be reconstituted with the recommended buffer immediately prior to use. Careful pipetting and dilution are essential for accurate quantification.
• Validation: The recombinant standard must be validated for use in your specific ELISA format. Commercial ELISA kits routinely use recombinant TIMP-1 as the calibration standard, demonstrating its suitability for quantitative assays.
• Compatibility: Ensure the recombinant TIMP-1 standard matches the epitope recognized by your ELISA antibodies. Most kits specify compatibility with human TIMP-1 and provide matched antibody pairs for optimal detection.
• Application: Recombinant TIMP-1 is explicitly recommended for use as an ELISA standard in multiple protocols and product datasheets.

Protocol best practices:

• Reconstitute the lyophilized standard as directed, typically with dilution buffer, and allow sufficient time for complete dissolution.
• Prepare serial dilutions to generate a standard curve covering the assay’s dynamic range (e.g., 62.5–4000 pg/mL).
• Run standards in parallel with samples to ensure accurate quantification.

Limitations:

• Recombinant standards are not recommended for bioassay applications unless specifically validated for those uses.
• Always confirm that your recombinant TIMP-1 preparation is suitable for ELISA calibration and not intended solely for other applications.

Summary Table: Recombinant Human TIMP-1 as ELISA Standard

In conclusion, recombinant human TIMP-1 is appropriate for use as a standard in ELISA quantification, provided it is properly formulated, validated, and compatible with your assay system.

Applications of Recombinant Human TIMP-1 in Published Research

Recombinant human TIMP-1 has been validated across a diverse range of applications in published research, reflecting its multifaceted biological roles.

Matrix Metalloproteinase Inhibition Studies

The primary application involves enzyme inhibition assays, where recombinant TIMP-1 demonstrates potent inhibitory activity against matrix metalloproteinases. The protein exhibits an IC₅₀ of 2.5-4 nM for inhibiting recombinant human MMP-2 cleavage of colorimetric peptide substrates, and an IC₅₀ of ≤34 ng/mL against activated mouse MMP-9. This inhibitory capacity has been extensively characterized in functional assays.

Immunological and Cellular Studies

Recombinant TIMP-1 has been applied in bioassay formats to investigate its immunomodulatory functions. Recent research demonstrates that TIMP-1 acts as an activator of MHC-I expression in myeloid dendritic cells, enhancing antigen presentation and CD8⁺ T cell activation. The protein has also been used to examine its role in cytokine signaling pathways and immune responses.

Molecular and Developmental Research

Studies have employed recombinant TIMP-1 to investigate its effects on cell proliferation and differentiation. Research utilizing lentiviral-mediated approaches has examined TIMP-1's negative regulatory role in osteogenic differentiation of human bone marrow-derived mesenchymal stem cells through Wnt/β-catenin signaling.

Analytical Applications

Recombinant TIMP-1 serves as a standard in Western blotting, ELISA, and immunoassay formats. The protein is particularly suitable for use as an ELISA standard when paired with matched antibody pairs, and has been validated for blocking assays and functional assays.

Quality Specifications

Recombinant TIMP-1 used in published research typically maintains very low endotoxin levels (<0.01 EU/μg), ensuring suitability for sensitive biological applications and minimizing confounding inflammatory responses in cell-based studies.

To reconstitute and prepare Recombinant Human TIMP-1 protein for cell culture experiments, dissolve the lyophilized protein in sterile, deionized water at a concentration of 100 μg/mL or higher. This stock solution can then be further diluted in your desired cell culture medium or buffer for experimental use.

Step-by-step protocol:

1. Preparation:

   • Allow the lyophilized TIMP-1 vial to reach room temperature before opening to minimize condensation.
   • Briefly centrifuge the vial (20–30 seconds) to collect all powder at the bottom.

2. Reconstitution:

   • Add sterile, deionized water (18 MΩ·cm recommended) to achieve a final concentration of at least 100 μg/mL.
   • Gently mix by swirling or inverting; avoid vigorous vortexing or foaming, as this can denature the protein.
   • Let the solution sit at room temperature for 10–15 minutes, gently mixing occasionally until fully dissolved.

3. Dilution:

   • Once dissolved, dilute the stock solution to your working concentration using sterile cell culture medium or buffer compatible with your assay.
   • For cell culture, phosphate-buffered saline (PBS, pH 7.4) is commonly used for further dilution.

4. Storage:

   • Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles.
   • Store aliquots at −20 °C or −80 °C for long-term storage; short-term storage at 2–8 °C is acceptable for up to one month.
   • For extended storage, adding 0.1% BSA can help stabilize the protein.

Additional notes:

• Do not reconstitute at concentrations above 1 mg/mL to avoid solubility issues.
• Confirm protein integrity by SDS-PAGE if needed.
• Avoid direct dissolution at elevated temperatures (e.g., 37 °C).
• If using for functional assays, ensure the buffer is compatible with TIMP-1 bioactivity and cell viability.

Summary Table:

This protocol ensures optimal solubility and bioactivity of recombinant TIMP-1 for cell culture applications.