Recombinant Mouse MAdCAM-1

Recombinant Mouse MAdCAM-1 is widely used in research to study immune cell trafficking, mucosal immunology, and inflammatory diseases due to its critical role as a ligand for integrin α4β7 and L-selectin, mediating lymphocyte homing to gut-associated lymphoid tissues.

Key reasons to use recombinant Mouse MAdCAM-1 in research applications:

• Modeling Lymphocyte Adhesion and Homing: MAdCAM-1 is essential for the adhesion and migration of lymphocytes, especially to the gut mucosa, by interacting with integrin α4β7 and L-selectin on lymphocytes. Recombinant protein enables controlled in vitro assays to dissect these interactions, such as adhesion, rolling, and transmigration assays.

• Bioactivity and Functional Assays: Recombinant MAdCAM-1 is validated for use in bioactivity assays, allowing researchers to study integrin-mediated signaling, test inhibitors, or screen for molecules that modulate lymphocyte trafficking. It is also used in flow-based adhesion assays to mimic physiological shear stress conditions.

• Inflammatory Disease Models: MAdCAM-1 is upregulated in inflammatory conditions (e.g., by TNF-α, IL-1β), making it a relevant target for studying diseases like inflammatory bowel disease (IBD). Recombinant protein can be used to evaluate therapeutic candidates that block MAdCAM-1 interactions or to model disease mechanisms in vitro.

• Reproducibility and Purity: Recombinant forms offer high purity (>95%) and batch-to-batch consistency, which are critical for reproducible experimental results.

• Versatility in Experimental Design: Recombinant MAdCAM-1 can be used in various formats, including as a substrate for cell adhesion, in ELISA-based binding assays, or as a capture reagent in flow cytometry and mass cytometry.

• Species-Specific Studies: Using mouse MAdCAM-1 is essential for studies involving mouse models, ensuring species compatibility and physiological relevance.

Typical applications include:

• Cell adhesion and migration assays
• Integrin-ligand binding studies
• Screening of therapeutic antibodies or small molecules targeting MAdCAM-1
• Modeling immune cell trafficking in gut-associated lymphoid tissue
• Investigating the molecular mechanisms of mucosal immunity and inflammation

In summary, recombinant Mouse MAdCAM-1 is a critical tool for dissecting the molecular basis of lymphocyte homing, studying mucosal immunology, and developing therapeutics for inflammatory diseases, with broad utility in both basic and translational research.

You can use recombinant Mouse MAdCAM-1 as a standard for quantification or calibration in your ELISA assays, provided the recombinant protein is properly validated and compatible with your assay system.

Key considerations:

• Recombinant protein as standard: Many commercial mouse MAdCAM-1 ELISA kits use recombinant Mouse MAdCAM-1 as the calibration standard to generate the standard curve for quantification. This is a common and accepted practice in quantitative ELISA assays for proteins where purified native protein is not readily available.

• Validation: Ensure the recombinant Mouse MAdCAM-1 you use is of high purity, correctly folded, and biologically active. It should match the epitope recognized by the capture and detection antibodies in your ELISA. Most kits specify that their standards are recombinant proteins, and recovery/linearity data are typically generated using recombinant Mouse MAdCAM-1 spiked into biological matrices.

• Carrier protein: Recombinant proteins may be supplied carrier-free or with stabilizers such as BSA. For ELISA standards, using a formulation with BSA can improve stability and reproducibility, but you should confirm that BSA does not interfere with your assay.

• Standard curve preparation: Follow the manufacturer's instructions for reconstitution and dilution of the recombinant standard. Prepare a serial dilution to cover the assay's dynamic range, and use the same buffer as your samples to minimize matrix effects.

• Assay compatibility: The recombinant Mouse MAdCAM-1 should be validated for use with your specific ELISA kit or custom assay. Some kits are designed to detect native protein only, so check the kit documentation to confirm compatibility.

Best practices:

• Run the recombinant standard in parallel with your samples on every plate.
• Confirm recovery and linearity by spiking recombinant Mouse MAdCAM-1 into your sample matrix.
• Use a standard curve generated from the recombinant protein to interpolate sample concentrations.

In summary, recombinant Mouse MAdCAM-1 is suitable as a standard for ELISA quantification if it is validated for your assay and matches the target protein's immunoreactivity. Always consult your assay protocol and validate the standard in your specific experimental context.

Recombinant Mouse MAdCAM-1 has been validated for several key applications in published research, primarily in studies of lymphocyte adhesion, migration, and inflammation. The most commonly reported applications include:

• Bioassays: Used to study the functional interaction between MAdCAM-1 and its ligands, such as integrin α4β7, in cell-based assays. These bioassays help elucidate the role of MAdCAM-1 in lymphocyte homing, immune cell recruitment, and inflammatory responses, particularly in models of inflammatory bowel disease (IBD), diabetes, and colitis.

• Adhesion Assays: Employed to measure the binding of lymphocytes (especially those expressing α4β7 integrin) to MAdCAM-1, often under static or flow conditions. These assays are critical for investigating the molecular mechanisms of leukocyte-endothelial interactions and extravasation in gut-associated lymphoid tissues.

• In Vivo Functional Studies: Recombinant MAdCAM-1 has been used in animal models to assess its role in leukocyte trafficking, intestinal inflammation, and disease progression. For example, blocking MAdCAM-1 in vivo reduces leukocyte extravasation and ameliorates intestinal inflammation in mouse models of colitis.

• Mass Cytometry (CyTOF) and Spatial Biology: Recent studies have validated recombinant MAdCAM-1 for advanced applications such as mass cytometry and spatial biology, enabling high-dimensional analysis of cell populations and tissue localization.

• Western Blotting: Used to detect and quantify MAdCAM-1 protein expression in tissue samples, often in the context of disease models or following experimental treatments.

• Vaccine Development: Recombinant MAdCAM-1 has been explored as a tool in vaccine research, particularly for its role in modulating immune cell trafficking and mucosal immunity.

• Receptor Occupancy Assays: Utilized to measure the binding of therapeutic antibodies or ligands to α4β7 integrin, using soluble MAdCAM-1 as a probe to detect free receptor sites.

Supporting Details:

• MAdCAM-1 is a critical adhesion molecule for lymphocyte homing to mucosal tissues, especially the gut, and is upregulated in inflammatory conditions such as IBD and colitis.
• Functional studies often use recombinant MAdCAM-1 to dissect the molecular basis of immune cell recruitment and to test therapeutic interventions targeting the MAdCAM-1/α4β7 axis.
• Adhesion assays with recombinant MAdCAM-1 are widely used to validate the specificity and efficacy of antibodies or small molecules that block lymphocyte trafficking.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed publications and are central to research in immunology, inflammation, and mucosal biology.

To reconstitute and prepare Recombinant Mouse MAdCAM-1 protein for cell culture experiments, dissolve the lyophilized protein in sterile PBS (pH 7.2–7.4) or sterile water to a stock concentration of 100 μg/mL (or as recommended by the supplier), then dilute to your working concentration in cell culture medium or buffer as needed.

Step-by-step protocol:

• Preparation:

  • Briefly centrifuge the vial to collect the lyophilized powder at the bottom before opening.
  • Use aseptic technique throughout to maintain sterility.

• Reconstitution:

  • Add sterile PBS (pH 7.2–7.4) or sterile water to the vial to achieve a stock concentration of 100 μg/mL (or as specified in the product datasheet).
  • Gently mix by pipetting up and down; do not vortex as this may denature the protein.
  • Allow the protein to dissolve completely at room temperature for several minutes.

• Aliquoting and Storage:

  • Aliquot the stock solution to avoid repeated freeze-thaw cycles.
  • Store aliquots at 2–8 °C for short-term use (up to one week), or at –20 °C to –80 °C for long-term storage (up to 12 months).
  • Use a manual defrost freezer and avoid repeated freeze-thaw cycles to preserve protein activity.

• Working Solution Preparation:

  • Dilute the stock solution to the desired working concentration in cell culture medium or assay buffer immediately before use.
  • For cell adhesion assays, typical coating concentrations are 10 μg/mL in PBS, applied to plates at 100 μL/well.
  • If required, supplement with carrier protein (e.g., 0.2–1% BSA) to stabilize the protein, especially at low concentrations.

Additional notes:

• Confirm the buffer composition and recommended concentration from the specific product datasheet, as formulations may vary depending on the expression system and purification method.
• Avoid vigorous mixing or vortexing, which can denature the protein.
• If the protein was originally lyophilized from PBS, reconstitute in PBS to maintain the original salt concentration; otherwise, use sterile water and dilute in PBS for working solutions.
• For functional assays, ensure endotoxin levels are sufficiently low (<0.1 EU/μg) for cell culture applications.

Summary of key points:

• Reconstitute in sterile PBS (pH 7.2–7.4) or sterile water to 100 μg/mL.
• Aliquot and store at –20 °C to –80 °C.
• Dilute to working concentration in cell culture medium or buffer before use.
• Avoid repeated freeze-thaw cycles and vigorous mixing.

These steps will ensure optimal preparation and stability of recombinant Mouse MAdCAM-1 protein for cell culture experiments.