Recombinant Human Cardiotrophin-1

Recombinant human cardiotrophin-1 (CT-1) is a valuable research tool with diverse applications across cardiovascular, immunological, and cellular biology research. Here are the key reasons to incorporate it into your experimental work:

Cardiovascular Research Applications

CT-1 is particularly well-suited for studying cardiac biology and pathophysiology. The protein induces cardiomyogenesis, cardiomyocyte growth and survival, and cardiomyocyte hypertrophy. It also promotes vascular remodeling of the heart, making it essential for investigating cardiac adaptation and remodeling mechanisms. Additionally, CT-1 enhances monocyte adhesion and migration by stimulating intercellular adhesion molecule-1 and CCL2 on human aortic endothelial cells, providing insights into vascular-immune interactions in cardiovascular disease.

Immunomodulatory Functions

CT-1 demonstrates significant immunomodulatory properties that extend its research utility beyond cardiac applications. The protein can activate monocytes and modulate cytokine production of activated CD4+ T-lymphocytes in vitro. Specifically, CT-1 causes concentration-dependent increases in tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) in monocytes. This makes it valuable for investigating immune activation mechanisms and the interplay between cardiac and immune systems, particularly in disease states like congestive heart failure where immunactivation occurs.

Bioassay and Functional Studies

Recombinant CT-1 is highly suitable for bioassay applications. The protein induces proliferation of TF-1 human erythroleukemia cells with an ED50 of 0.5-2 ng/mL, providing a quantifiable readout for functional studies. This bioactivity makes it ideal for dose-response experiments and validation of signaling pathway activation.

Receptor Signaling Research

CT-1 signals through a heterodimeric receptor formed by leukemia inhibitory factor receptor beta (LIFR) and gp130, allowing researchers to investigate gp130-mediated signaling pathways. This is particularly relevant for studying cytokine signaling in the IL-6 family, as CT-1 belongs to this cytokine group.

Clinical Relevance and Biomarker Studies

Elevated CT-1 plasma levels are associated with multiple cardiovascular pathologies including hypertensive heart disease, aortic stenosis, coronary artery disease, and dilated cardiomyopathy. Using recombinant CT-1 in research helps elucidate the mechanisms underlying these disease states and validates CT-1 as a biomarker for hypertension and cardiovascular risk associated with diabetes.

Production and Quality Considerations

High-purity recombinant CT-1 preparations (>95% purity) are available with reproducible results in bioactivity assays. The protein is available in both carrier-containing and carrier-free formulations, allowing you to select the appropriate format for your specific experimental requirements. For cell or tissue culture applications, carrier-containing formulations are generally recommended, while carrier-free versions are preferred for ELISA standards and certain sensitive applications.

Yes, recombinant Human Cardiotrophin-1 can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated for your specific assay system. Recombinant CT-1 is commonly supplied as the standard in commercial ELISA kits, and its use for generating standard curves is well-established in the literature and protocols.

Supporting details:

• Standard Curve Preparation: ELISA kits for Human Cardiotrophin-1 routinely use recombinant CT-1 to prepare standard curves, with concentrations spanning the assay’s dynamic range (e.g., 16.46–12,000 pg/mL). The recombinant protein is reconstituted and serially diluted to generate calibration points for quantification.

• Assay Validation: The recombinant standard is validated for linearity, recovery, and specificity in various sample matrices (serum, plasma, cell culture supernatants). Linearity and percent recovery data confirm that recombinant CT-1 behaves comparably to endogenous protein in these matrices.

• Specificity: Sandwich ELISA formats employing matched antibody pairs are designed to recognize both natural and recombinant Human Cardiotrophin-1, ensuring accurate quantification regardless of the protein source.

• Best Practices:

  • Ensure the recombinant CT-1 standard is of high purity and correctly folded, as improper folding or contaminants may affect assay accuracy.
  • Validate the standard in your specific assay system, especially if using a custom or in-house ELISA, to confirm comparable reactivity and recovery.
  • Follow the manufacturer’s instructions for reconstitution, dilution, and storage to maintain protein integrity.

• Limitations: Some ELISA kits may note that their calibration is optimized for native CT-1 and recommend caution when quantifying recombinant protein in unknown samples. However, for standard curve generation and calibration, recombinant CT-1 is the accepted standard.

Summary Table: Use of Recombinant Human Cardiotrophin-1 as ELISA Standard

In conclusion, recombinant Human Cardiotrophin-1 is suitable and routinely used as a standard for ELISA quantification and calibration, provided it is validated for your assay conditions and sample types.

Recombinant Human Cardiotrophin-1 (CT-1) has been validated in published research for a variety of applications, primarily in the context of cardiovascular biology, cell signaling, and disease modeling. Key applications supported by scientific literature include:

• Bioassay: CT-1 is widely used in bioassays to study its effects on cell proliferation, survival, and signaling. For example, it induces proliferation of TF-1 human erythroleukemia cells and is used to assess activity in cell-based assays (e.g., ED50 determination).

• Cardiomyocyte Hypertrophy and Survival: CT-1 has been shown to induce hypertrophy and promote survival of cardiac myocytes, both in vitro and in vivo. It is used to study cardiac growth, protection against ischemic injury, and apoptosis.

• Cardioprotection: Studies have demonstrated that CT-1 protects cardiac cells from stressors such as heat shock, ischemia, and hypoxia, making it a tool for investigating cardioprotective mechanisms.

• Vascular Remodeling and Endothelial Cell Function: CT-1 enhances monocyte adhesion and migration by stimulating intercellular adhesion molecule-1 and CCL2 on human aortic endothelial cells, and is used to study vascular inflammation and remodeling.

• Western Blot: CT-1 has been detected and analyzed using Western blot in studies of its expression and regulation, for example in choroid plexus and cerebrospinal fluid.

• Biomarker Research: CT-1 plasma levels are studied as a biomarker for cardiovascular diseases such as hypertensive heart disease, aortic stenosis, coronary artery disease, and dilated cardiomyopathy.

• Cancer and Tumor-Stroma Interactions: CT-1 (CTF1) is implicated in tumor-stroma interactions, fibroblast activation, and cancer metastasis, and is used in studies of cancer biology and autophagy.

• Adipose Tissue Biology: CT-1 is expressed in adipose tissue and is studied in the context of metabolic regulation and adipose tissue dysfunction.

• Signal Transduction Studies: CT-1 is used to investigate signaling pathways, particularly those involving the gp130/LIF receptor complex and downstream effects such as induction of heat shock proteins.

These applications are supported by peer-reviewed studies and are commonly cited in the context of recombinant CT-1 use in research.

To reconstitute and prepare Recombinant Human Cardiotrophin-1 (CT-1) protein for cell culture experiments, follow these best-practice steps:

1. Centrifuge the vial briefly before opening to ensure all lyophilized protein is at the bottom.

2. Reconstitution buffer:

   • Use 20 mM Tris, pH 8.0 as the reconstitution buffer.
   • Alternatively, some protocols recommend sterile 4 mM HCl with at least 0.1% BSA or HSA as a carrier protein, especially if the protein is highly hydrophobic or prone to aggregation.
   • For acidic reconstitution (e.g., 0.1 M acetate buffer, pH 4), adjust to neutral pH after dissolution if required for your application.

3. Protein concentration:

   • Reconstitute to a final concentration of 0.1–1.0 mg/mL.
   • Gently mix by pipetting or swirling; avoid vigorous shaking or vortexing to prevent protein denaturation.

4. Further dilution:

   • For working concentrations, dilute the reconstituted stock in aqueous buffer containing 0.1–1% carrier protein (e.g., BSA or HSA) to minimize adsorption and stabilize the protein.
   • Use sterile PBS or cell culture medium as the diluent, depending on your assay requirements.

5. Aliquoting and storage:

   • Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles.
   • Store aliquots at 2–8°C for up to one week for short-term use.
   • For long-term storage, keep at –20°C or –70°C; avoid multiple freeze-thaw cycles to preserve activity.

6. General notes:

   • Always use sterile technique to prevent contamination.
   • If the protein was reconstituted in acidic buffer, ensure the final working solution is compatible with cell culture (neutral pH, isotonic).
   • The ED50 for biological activity (e.g., TF-1 cell proliferation) is typically 0.5–2 ng/mL; optimize dosing for your specific cell type and assay.

Summary protocol example:

1. Briefly centrifuge the vial.2. Add sterile 20 mM Tris, pH 8.0, to achieve 0.1–1.0 mg/mL.3. Gently mix until fully dissolved.4. Dilute to working concentration in sterile PBS or medium with 0.1–1% BSA.5. Aliquot and store at –20°C or –70°C for long-term use; 2–8°C for up to one week.

Do not use vigorous agitation or repeated freeze-thaw cycles, and always include a carrier protein in dilutions to maintain stability and bioactivity.