Recombinant Human VCAM-1

Recombinant Human VCAM-1 is widely used in research to study cell adhesion, migration, and inflammatory processes, especially those involving leukocyte-endothelial interactions and vascular biology. Its use is critical for dissecting mechanisms of immune cell trafficking, vascular inflammation, and related pathologies.

Key scientific reasons to use recombinant VCAM-1 include:

• Modeling Leukocyte-Endothelial Adhesion: VCAM-1 mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils to activated endothelial cells, a central event in inflammation and immune response.
• Studying Inflammatory Disease Mechanisms: VCAM-1 is implicated in the development of atherosclerosis, rheumatoid arthritis, and other inflammatory diseases, making it a valuable tool for investigating disease pathways and potential therapeutic targets.
• Functional Assays: Recombinant VCAM-1 can be used in bioassays to measure cell adhesion, migration, and transmigration, as well as in ELISA as a standard for quantification and calibration.
• Drug Discovery and Screening: VCAM-1 is a promising drug target for cardiovascular and inflammatory diseases, and recombinant protein enables screening of inhibitors or antibodies that modulate its function.
• Immunological Applications: It serves as a positive control in Western blotting, immunoprecipitation, and other immunological assays.

Additional relevant information:

• Protein Quality: Recombinant VCAM-1 is typically produced with high purity and low endotoxin levels, ensuring suitability for sensitive cell-based assays.
• Versatility: It is validated for use in both basic research and translational studies, including cell culture, tissue assays, and animal models.
• Clinical Relevance: VCAM-1 levels are prognostic in certain cancers and cardiovascular conditions, and recombinant protein is used in diagnostic assay development.

In summary, using recombinant human VCAM-1 enables precise, reproducible investigation of cell adhesion and inflammation, supports assay standardization, and facilitates translational research into vascular and immune-related diseases.

Yes, recombinant human VCAM-1 can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated and matches the form of VCAM-1 detected by your assay.

Key considerations and supporting details:

• Assay Compatibility: Most commercial human VCAM-1 ELISA kits use recombinant human VCAM-1 as the standard for generating the calibration curve. These kits are designed to recognize both endogenous (native) and recombinant forms of VCAM-1, ensuring accurate quantification in biological samples.

• Validation: It is essential to confirm that the recombinant VCAM-1 standard is structurally and immunologically similar to the VCAM-1 present in your samples. This includes matching the isoform, glycosylation status, and sequence region (e.g., extracellular domain). The standard should be validated for recovery and linearity in your specific sample matrix (serum, plasma, cell culture supernatant).

• Standard Curve Preparation: Prepare a dilution series of the recombinant VCAM-1 standard according to your ELISA protocol, typically covering the assay’s detection range (e.g., 0.79–50 ng/mL or higher). Use freshly prepared standard solutions for each experiment to ensure accuracy.

• Recovery and Linearity: Commercial kits report high recovery rates (typically 88–105%) when recombinant VCAM-1 is spiked into serum or plasma, indicating suitability for quantification. Linearity of dilution should also be confirmed to ensure accurate interpolation of sample concentrations.

• Documentation: Always refer to your ELISA kit’s technical manual for specific instructions regarding standard preparation and validation. Some kits may specify the exact recombinant VCAM-1 sequence used as the standard.

In summary, recombinant human VCAM-1 is widely accepted as a standard for ELISA quantification, but ensure it is validated for your assay and sample type for reliable results.

Recombinant Human VCAM-1 has been validated for a broad range of applications in published research, primarily in studies of cell adhesion, migration, inflammatory processes, and vascular biology. Key validated applications include:

• Cell adhesion assays: Used to study leukocyte-endothelial interactions, particularly the adhesion of lymphocytes, monocytes, eosinophils, and basophils to endothelial cells.
• Cell migration assays: Employed to investigate mechanisms of leukocyte transmigration and homing, including in models of inflammation and cancer metastasis.
• Bioassays: Utilized in functional studies to assess the biological activity of VCAM-1, such as its role in osteoclast differentiation, hematopoietic progenitor mobilization, and T cell development from pluripotent stem cells.
• ELISA (Enzyme-Linked Immunosorbent Assay): Used as a standard or positive control for quantification and calibration in immunoassays.
• Western blotting: Applied as a positive control for protein detection and validation.
• Immunoprecipitation: Used in immunological experiments to isolate and study VCAM-1 or its binding partners.
• Imaging and diagnostic research: Incorporated in the development of targeted imaging agents for noninvasive detection of atherosclerotic lesions and as a biomarker in inflammatory and cancer studies.

Published research examples:

• Cancer metastasis: VCAM-1 has been used to study its role in promoting osteolytic bone metastasis and as a target for therapeutic intervention in breast cancer models.
• Hematopoietic differentiation: VCAM-1, in combination with DLL4, has been shown to enhance the emergence of T cell–competent hematopoietic progenitors from human pluripotent stem cells.
• Inflammatory disease models: VCAM-1 is validated as a biomarker and functional mediator in diseases such as atherosclerosis, asthma, rheumatoid arthritis, and inflammatory bowel disease.

Summary of validated applications:

These applications demonstrate the versatility of recombinant human VCAM-1 in both basic and translational research.

To reconstitute and prepare Recombinant Human VCAM-1 protein for cell culture experiments, follow these best-practice steps based on current protocols and manufacturer recommendations:

1. Centrifuge the vial:
Briefly centrifuge the lyophilized protein vial before opening to ensure all material is at the bottom.

2. Reconstitution:

• Use sterile, deionized water or sterile PBS (without calcium or magnesium unless specified) to reconstitute the protein.
• A common reconstitution concentration is 1 mg/mL. Some protocols recommend not going below 100 μg/mL to maintain protein stability.
• Gently dissolve the protein by swirling or gentle pipetting. Do not vortex or mix vigorously, as this can denature the protein.

Example protocol:

- Add the appropriate volume of sterile water or PBS to achieve the desired concentration (e.g., 1 mg/mL).- Allow the vial to sit at room temperature for 10–15 minutes.- Gently mix by inverting or pipetting up and down.

3. Aliquoting and Storage:

• Aliquot the reconstituted protein into small volumes to avoid repeated freeze-thaw cycles, which can degrade the protein.
• Store aliquots at –20°C to –80°C for long-term storage. For short-term use (up to 1 week), store at 2–8°C under sterile conditions.
• Avoid repeated freeze-thaw cycles.

4. Preparation for Cell Culture:

• Before use, dilute the protein to the working concentration using cell culture-grade buffer (e.g., PBS or serum-free medium).
• Typical coating concentrations for cell adhesion assays are 5–10 μg/mL in PBS, applied to tissue culture plates and incubated at 4°C overnight or at room temperature for 1–2 hours.
• After coating, wash wells with PBS to remove unbound protein before adding cells.

5. Additional Notes:

• If the protein has a tag (e.g., Fc or His), ensure compatibility with your assay and detection system.
• For functional assays, confirm the protein is endotoxin-free if required for sensitive cell types.

Summary Table: Key Steps and Conditions

Always consult the specific datasheet for your VCAM-1 preparation, as formulation and recommended protocols may vary depending on the expression system and tags used.