Recombinant Mouse VCAM-1

Recombinant Mouse VCAM-1 is widely used in research applications because it enables precise modeling and investigation of cell adhesion, migration, and inflammatory processes, particularly those involving immune cell interactions and vascular biology.

Key scientific reasons to use recombinant mouse VCAM-1 include:

• Modeling Cell Adhesion and Migration: VCAM-1 is a critical adhesion molecule expressed on activated endothelial cells, mediating leukocyte binding and transendothelial migration during inflammation. Recombinant VCAM-1 allows controlled in vitro studies of these processes, such as immune cell trafficking, endothelial activation, and migration assays.

• Disease Mechanism Studies: VCAM-1 is implicated in the pathogenesis of various diseases, including atherosclerosis, rheumatoid arthritis, asthma, inflammatory bowel disease, and cancer metastasis. Recombinant VCAM-1 enables mechanistic studies of these diseases, including modeling vascular inflammation and immune cell infiltration.

• Drug Target Validation and Screening: VCAM-1 is a validated drug target for several inflammatory and vascular diseases. Recombinant VCAM-1 can be used in bioassays to screen and characterize inhibitors, antibodies, or small molecules that modulate VCAM-1-mediated adhesion or signaling.

• Angiogenesis Research: VCAM-1, especially its Ig-like domain 6, plays a role in TNFα-induced angiogenesis. Recombinant VCAM-1 or its domains can be used to dissect angiogenic pathways and test therapeutic candidates targeting vascular remodeling.

• Immunological Assays: Recombinant VCAM-1 is suitable as a standard or positive control in ELISA, Western blotting, immunoprecipitation, and flow cytometry, facilitating quantification and detection of VCAM-1 or its binding partners.

• Stem Cell and Differentiation Studies: VCAM-1 is involved in hematopoietic progenitor emergence and differentiation. Recombinant VCAM-1 supports research into stem cell biology and immune cell development.

• Imaging and Biomarker Development: VCAM-1 is a biomarker for vascular inflammation and can be used to develop imaging agents or molecular probes for detecting inflamed endothelium in vivo.

Best practices for using recombinant mouse VCAM-1:

• Select the appropriate formulation (e.g., carrier-free or with BSA) depending on your application, such as cell culture, ELISA, or bioassays.
• Validate activity and purity for your specific experimental needs, especially for quantitative or functional assays.

In summary, recombinant mouse VCAM-1 is a versatile tool for dissecting vascular and immune mechanisms, validating therapeutic targets, and developing diagnostic assays in preclinical research.

Yes, recombinant mouse VCAM-1 can be used as a standard for quantification and calibration in ELISA assays. The search results confirm that commercial ELISA kits are specifically designed to recognize and measure both natural and recombinant forms of the protein.

Key Considerations for Using Recombinant VCAM-1 Standards

Recognition and Compatibility

The antibodies used in sandwich ELISA formats are engineered to bind epitopes present on both native and recombinant VCAM-1 molecules. This cross-reactivity ensures that recombinant standards will generate reliable calibration curves that accurately reflect the binding characteristics of the detection system.

Standard Preparation

When preparing recombinant VCAM-1 standards, follow these best practices:

• Dissolve lyophilized standards in the appropriate diluent buffer provided with your assay kit within 15 minutes before use
• Prepare serial dilutions using a buffered protein base to maintain protein stability
• Create multiple concentration points (typically 7-8 dilution points) spanning your expected detection range
• Use fresh standard solutions for each experiment to ensure accuracy

Performance Characteristics

Recombinant standards typically achieve excellent precision metrics. Intra-assay precision (within a single plate) generally yields coefficient of variation (CV) values below 8%, while inter-assay precision (between separate assays) typically remains below 10%. These precision levels are suitable for most quantitative applications.

Detection Range and Sensitivity

The sensitivity of your assay will depend on the specific antibody pair and kit configuration used. Typical minimum detectable doses for mouse VCAM-1 range from approximately 0.12 to 0.3 ng/mL, depending on the assay format. Ensure your recombinant standard concentration range encompasses both your expected sample values and provides adequate spacing for accurate curve fitting.

Sample Matrix Considerations

When validating your recombinant standard calibration, test recovery in relevant biological matrices (serum, plasma, cell culture supernatant) to confirm that the standard curve accurately predicts analyte concentration in your actual samples.

Recombinant Mouse VCAM-1 has been validated in published research for several key applications, primarily in bioassays involving cell adhesion, migration, and signaling, as well as in molecular imaging, affinity measurements, and as a standard in analytical assays.

Key validated applications include:

• Cell-based bioassays:
  Used to study leukocyte-endothelial interactions, T cell migration, and integrin-mediated signaling. For example, it has been employed to assess T cell migration across endothelial barriers, integrin signaling in immune cells, and the emergence of T cell–competent hematopoietic progenitors from stem cells.

• Molecular imaging and tracer validation:
  Recombinant mouse VCAM-1 has been used as a target antigen for the development and validation of imaging agents, such as radiolabeled nanobodies, for noninvasive detection of VCAM-1 expression in vivo (e.g., SPECT/CT imaging of atherosclerotic plaques).

• Affinity and binding studies:
  Utilized in surface plasmon resonance (SPR) and flow cytometry to characterize the binding affinity and specificity of antibodies, nanobodies, and peptides targeting VCAM-1.

• Analytical standards and controls:
  Applied as a standard in ELISA and other immunoassays to quantify VCAM-1 levels or validate antibody specificity.

• Protein characterization:
  Used in SDS-PAGE, HPLC, and mass spectrometry for quality control and validation of recombinant protein preparations.

• Functional studies:
  Employed in research on inflammatory diseases, atherosclerosis, and immune cell trafficking to model VCAM-1-mediated processes.

Summary Table of Validated Applications

These applications are well-supported by published research and product validation data, demonstrating the versatility of recombinant mouse VCAM-1 in both basic and translational studies.

To reconstitute and prepare Recombinant Mouse VCAM-1 protein for cell culture experiments, briefly centrifuge the vial to collect the lyophilized protein, then add sterile water or buffer to achieve the desired concentration (typically 0.1–0.5 mg/mL), and gently mix until fully dissolved. Use sterile technique throughout to prevent contamination.

Detailed Protocol:

• Centrifugation: Before opening the vial, briefly centrifuge to ensure all lyophilized protein is at the bottom.
• Reconstitution: Add sterile, deionized water or sterile PBS (pH 7.4) to the vial. The recommended concentration for reconstitution is usually between 0.1–0.5 mg/mL. Gently mix by pipetting or swirling; avoid vigorous agitation to prevent protein denaturation.
• Dissolution: Allow the protein to dissolve completely. This may take several minutes at room temperature. Do not vortex.
• Aliquoting: Once dissolved, aliquot the solution to minimize freeze-thaw cycles, which can degrade protein activity.
• Storage: Store aliquots at -20°C to -70°C for long-term use. After reconstitution, the protein can be stored at 2–8°C for up to one month under sterile conditions, or at -20°C to -70°C for up to three months. Avoid repeated freeze-thaw cycles.
• Endotoxin Consideration: Confirm endotoxin levels are suitable for cell culture (typically <0.1 EU/μg protein).
• Preparation for Cell Culture: If adding directly to cell culture, dilute the reconstituted protein in sterile cell culture medium to the desired working concentration. For coating plates, dilute to 10 μg/mL in PBS or coating buffer, add 100 μL/well, and incubate at room temperature for 1 hour.

Additional Notes:

• If the protein is formulated with stabilizers (e.g., trehalose, mannitol, or glycerol), ensure compatibility with your cell culture system.
• For functional assays (e.g., cell adhesion), verify biological activity post-reconstitution using a relevant assay.
• Always consult the specific product datasheet for recommended buffer and concentration, as formulations may vary.

Summary Table:

This protocol ensures optimal solubility, stability, and biological activity of recombinant mouse VCAM-1 for cell culture applications.