Recombinant Human Omentin

Recombinant human omentin (also known as intelectin-1) offers substantial scientific value across multiple research applications due to its diverse biological functions and therapeutic potential.

Key Biological Functions

Omentin is a multifunctional protein with well-characterized roles in metabolic regulation and cellular protection. The protein functions as a visceral adipose tissue-derived adipokine that enhances insulin-stimulated glucose uptake in adipocytes, making it particularly valuable for metabolic research. Beyond glucose metabolism, omentin activates AMP-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS) phosphorylation in endothelial cells, and it functions as a vasodilator.

Research Applications

Metabolic and Endocrine Research

Omentin serves as a critical biomarker and functional protein for investigating insulin sensitivity and metabolic dysfunction. Elevated omentin expression correlates with improved disease outcomes in type 2 diabetes mellitus, polycystic ovary syndrome, and metabolic dysfunction-associated steatotic liver disease. The protein's ability to modulate insulin signaling through protein kinase B (Akt) activation makes it essential for metabolic pathway studies.

Cardiovascular Research

Recombinant omentin demonstrates protective roles following cardiovascular injury by limiting fibrosis, cardiac hypertrophy, and carotid artery intimal hyperplasia. It inhibits smooth muscle cell proliferation, migration, and calcification while promoting revascularization, making it valuable for cardiovascular disease modeling and therapeutic development.

Neuroprotection Studies

Recombinant human omentin exerts neuroprotection against hypoxia/reoxygenation injury through activation of the GAS6/Axl signaling pathway. Treatment with recombinant omentin increases cell viability, decreases reactive oxygen species generation and lactate dehydrogenase release, and reduces apoptotic rates in neuronal models, positioning it as a candidate for ischemia-reperfusion injury research.

Inflammatory and Immunological Research

Omentin downregulates inflammatory cytokine expression including TNF-α and IFN-γ while reducing oxidative stress. The protein increases anti-inflammatory cytokine IL-4 production and decreases pro-inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α, making it valuable for inflammation-related research.

Technical Advantages

Recombinant omentin is available in multiple expression systems and formats suitable for various experimental approaches, including Western blotting and functional assays. Quality-controlled preparations ensure protein activity verification through lot-specific assays, supporting reproducible research outcomes.

Yes, recombinant human Omentin can be used as a standard for quantification or calibration in ELISA assays, provided it is properly characterized and compatible with your assay system.

Supporting details:

• Recombinant proteins are commonly used as ELISA standards when purified native protein is unavailable or impractical to obtain. Many commercial ELISA kits for Omentin/Intelectin-1 use recombinant human Omentin as the standard for generating calibration curves.
• The recombinant standard should be well-characterized, with a known concentration and purity, and ideally matched to the sequence and post-translational modifications of the native analyte as closely as possible.
• When preparing your standard curve, ensure the recombinant Omentin is reconstituted and diluted according to best practices to minimize errors, especially during large dilution steps. It is recommended to value-assign the standard concentration based on measurement in ELISA rather than relying solely on the mass stated on the vial.
• The standard curve should cover the expected range of Omentin concentrations in your samples, typically from low ng/mL to several hundred ng/mL, depending on your assay sensitivity and sample type.
• If your ELISA kit or protocol specifies a particular form or source of Omentin (e.g., specific amino acid sequence, glycosylation state), ensure your recombinant standard matches these specifications for optimal accuracy.

Best practices:

• Use a recombinant Omentin standard that is validated for ELISA applications and, if possible, sourced from the same organism and expression system as your assay's antibodies.
• Prepare and store the standard according to manufacturer or protocol instructions, as improper handling can affect protein stability and quantification accuracy.
• Always run the standard curve in parallel with your samples and use curve-fitting models appropriate for your assay format (e.g., 4-parameter logistic regression).

Limitations:

• Minor differences in immunoreactivity or post-translational modifications between recombinant and native Omentin may affect quantification accuracy, but this is generally acceptable for research use.
• For diagnostic or clinical applications, additional validation may be required to ensure equivalence between recombinant and native standards.

In summary, recombinant human Omentin is suitable as a standard for ELISA quantification, provided it is properly validated and handled according to best practices.

Recombinant human omentin has been validated for several applications in published research, primarily in the context of metabolic, cardiovascular, and inflammatory studies.

Validated applications include:

• Western blotting: Recombinant human omentin has been directly validated for use in Western blotting to detect protein expression and signaling pathway activation in cell and tissue lysates.
• Functional cell-based assays: Omentin has been used to study its effects on cellular functions, such as:
  • Modulation of macrophage activity: Including suppression of inflammatory cytokine expression, reduction of lipid loading, and inhibition of apoptosis in macrophages.
  • Cardiomyocyte function: Recombinant omentin has been used to assess its ability to reverse contractile dysfunction and restore insulin-stimulated Akt phosphorylation in primary cardiomyocytes exposed to diabetic conditions.
  • Glucose uptake assays: Omentin has been shown to enhance insulin-stimulated glucose uptake in human adipocytes, often measured by radiolabeled glucose or fluorescent analog uptake assays.
  • Endothelial cell assays: Omentin has been used to suppress adhesion molecule expression (ICAM-1, VCAM-1) and reduce monocyte adhesion to endothelial cells.
• Immunoprecipitation and binding studies: Recombinant omentin has been used in co-immunoprecipitation and confocal microscopy to confirm its binding to integrin receptors (αvβ3, αvβ5).
• Immunohistochemistry and immunofluorescence: Used to localize omentin and assess its effects on tissue inflammation and cell infiltration in animal models.
• Bioassays in animal models: Recombinant omentin has been administered in vivo (e.g., via minipump infusion) to study its effects on atherosclerotic plaque stability, collagen content, and necrotic core formation in mouse models.

Additional research applications:

• SDS-PAGE: For protein purity and size assessment.
• ELISA and other immunoassays: While not always explicitly stated for recombinant protein, omentin is commonly measured in biological samples using immunoassays, and recombinant protein is often used as a standard or control.

Summary Table:

Key research areas:

• Metabolic regulation (insulin sensitivity, glucose uptake)
• Cardiovascular protection (atherosclerosis, cardiac function)
• Anti-inflammatory effects (macrophage and endothelial cell modulation)

If you need protocols or more detail on a specific application, please specify the assay or research context.

To reconstitute and prepare Recombinant Human Omentin protein for cell culture experiments, dissolve the lyophilized protein in sterile water at a concentration of ≥100 μg/mL (commonly 500 μg/mL is recommended), then dilute to your desired working concentration using cell culture-compatible buffer or medium.

Step-by-step protocol:

1. Centrifuge the vial briefly before opening to ensure all lyophilized powder is at the bottom.
2. Add sterile, endotoxin-free water directly to the vial to achieve the recommended stock concentration (e.g., 500 μg/mL, or at least 100 μg/mL).
3. Gently mix by pipetting up and down or by gentle swirling. Do not vortex, as vigorous agitation can denature the protein or cause foaming.
4. Allow the protein to dissolve at room temperature for 15–30 minutes with gentle agitation.
5. Ensure complete dissolution by gently washing down the sides of the vial with the solution.
6. Aliquot the stock solution into small volumes to avoid repeated freeze-thaw cycles.
7. Store aliquots at 4°C for short-term use (up to one week), or at –20°C to –80°C for long-term storage. For long-term storage, adding a carrier protein such as 0.1% BSA or HSA is recommended to enhance stability.
8. For cell culture experiments, dilute the stock solution into your cell culture medium or buffer immediately before use. If further dilution is needed, use PBS or medium containing at least 0.1% BSA to minimize adsorption and loss of protein activity.

Additional best practices:

• Avoid repeated freeze-thaw cycles, as these can degrade the protein.
• Filter the final working solution through a 0.2 μm sterile filter if sterility is required for cell culture.
• Confirm the absence of endotoxin if your cell type is sensitive.
• Always wear appropriate PPE and handle under sterile conditions.

Summary Table:

This protocol ensures optimal solubility, stability, and bioactivity of recombinant human Omentin for cell culture applications.