Recombinant Human VCAM-1

Recombinant Human VCAM-1 is used in research to model and investigate cell adhesion, migration, and inflammatory processes, particularly those involving leukocyte-endothelial interactions. It is a critical tool for studying diseases such as atherosclerosis, cancer, rheumatoid arthritis, asthma, and transplant rejection, where VCAM-1-mediated pathways play a central role.

Key reasons to use Recombinant Human VCAM-1 in research applications:

• Cell Adhesion and Migration Studies: VCAM-1 is an immunoglobulin-like adhesion molecule expressed on activated endothelial cells, mediating the adhesion and transmigration of leukocytes (lymphocytes, monocytes, eosinophils, basophils) across the endothelium. Recombinant VCAM-1 enables controlled in vitro assays to dissect these processes, such as adhesion assays and transmigration models.

• Inflammation and Disease Modeling: VCAM-1 is upregulated in response to inflammatory cytokines and is implicated in the pathogenesis of atherosclerosis, cancer metastasis, rheumatoid arthritis, and asthma. Using recombinant VCAM-1 allows researchers to study its role in these diseases, test inhibitors, and explore therapeutic interventions.

• Signal Transduction Research: VCAM-1 engagement activates downstream signaling pathways (e.g., Ca²⁺, Rac1, NOX2, ROS, MMPs, PKCα, PTP1B), which can be studied using recombinant protein in cell-based assays.

• Bioassays and ELISA Standards: Recombinant VCAM-1 is used as a standard in ELISA for quantifying soluble VCAM-1 in biological fluids, and as a positive control in Western blotting, immunoprecipitation, and other immunological assays.

• Therapeutic Target Validation: Recombinant VCAM-1 is essential for screening and characterizing antibodies, peptides, or small molecules that block VCAM-1 interactions, supporting drug discovery for inflammatory and cardiovascular diseases.

• Cancer Research: Elevated VCAM-1 expression is observed in several cancers, and recombinant VCAM-1 is used to study tumor cell adhesion, migration, and the tumor microenvironment.

• Technical Versatility: The protein is validated for a wide range of applications, including cell culture, bioassays, and as a reagent in translational studies.

In summary, Recombinant Human VCAM-1 is a versatile reagent for dissecting molecular mechanisms of cell adhesion, inflammation, and disease progression, as well as for developing and validating therapeutic strategies targeting VCAM-1-mediated pathways.

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

Key considerations and best practices:

• Form Consistency: The recombinant VCAM-1 standard should match the isoform and post-translational modifications (e.g., glycosylation) of the VCAM-1 present in your samples, as differences can affect antibody recognition and quantification accuracy.
• Validation: Confirm that your ELISA detects both natural and recombinant VCAM-1 equivalently. Many commercial ELISA kits use recombinant human VCAM-1 as the standard and validate that the assay recognizes both forms.
• Standard Curve Preparation: Prepare a dilution series of the recombinant VCAM-1 standard according to your assay protocol, typically covering the expected concentration range in your samples (e.g., 0.78–50 ng/mL or as specified by your kit).
• Parallelism: Assess parallelism between the standard curve and serially diluted samples to ensure accurate quantification across matrices.
• Documentation: Record the source, lot, and concentration of your recombinant standard for reproducibility and traceability.

Protocol Example:

• Reconstitute the lyophilized recombinant VCAM-1 standard as instructed.
• Prepare serial dilutions to generate a standard curve (e.g., 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 ng/mL).
• Run standards in duplicate or triplicate alongside your samples.
• Calculate sample concentrations by interpolating from the standard curve.

Limitations:

• If your samples contain a different VCAM-1 isoform or are heavily glycosylated compared to the recombinant standard, quantification may be less accurate unless the assay is specifically validated for those forms.
• Always verify that the recombinant standard is compatible with your specific ELISA antibodies and detection system.

Summary:
Recombinant human VCAM-1 is widely used as a standard in ELISA assays for quantification, but proper validation and matching to the assay’s detection characteristics are essential for accurate results.

Recombinant Human VCAM-1 has been validated for a broad range of applications in published research, primarily in studies of cell adhesion, immunological assays, cell signaling, and disease modeling.

Key validated applications include:

• Cell adhesion assays: Recombinant VCAM-1 is widely used to study the adhesion of leukocytes (e.g., lymphocytes, monocytes, eosinophils, basophils) to endothelial cells or to immobilized VCAM-1, modeling physiological and pathological cell trafficking.
• ELISA (Enzyme-Linked Immunosorbent Assay): It serves as a standard or capture antigen for quantifying VCAM-1 in biological samples, including serum, plasma, and cell culture supernatants.
• Bioassays: Used to assess the functional activity of VCAM-1 in cell migration, adhesion, and signaling, including studies on hematopoietic progenitor mobilization, leukocyte transmigration, and modulation of immune cell behavior.
• Western blotting: Employed as a positive control to validate antibody specificity and protein detection.
• Immunoprecipitation: Used to pull down VCAM-1 or VCAM-1-interacting proteins from cell lysates.
• Imaging and in vivo targeting: Recombinant VCAM-1 has been used in the development of imaging agents (e.g., radiolabeled nanobodies or antibodies) for noninvasive detection of VCAM-1 expression in disease models, such as atherosclerosis and cancer metastasis.
• Cell sorting and capture: Functionalized devices or chips coated with recombinant VCAM-1 have been used to capture VCAM-1–positive cells or extracellular vesicles for downstream analysis.
• Stem cell and differentiation assays: VCAM-1 has been used as a substrate or co-factor to enhance differentiation of human pluripotent stem cells into hematopoietic and T cell lineages, particularly in synergy with other signaling molecules like DLL4.
• Mass cytometry and spatial biology: Applied in advanced cytometric and spatial profiling platforms for multiplexed detection and localization of VCAM-1 in tissues.

These applications demonstrate the versatility of recombinant human VCAM-1 in both basic research (e.g., cell biology, immunology, stem cell differentiation) and translational studies (e.g., biomarker quantification, disease modeling, imaging agent development).

If you require protocols or more detailed references for a specific application, please specify the intended use.

Reconstitution Protocol

Initial Preparation

Before opening the vial, centrifuge it briefly to ensure all lyophilized protein settles to the bottom. This prevents loss of material during opening.

Add sterile, deionized water to reconstitute the protein. The standard reconstitution concentration is 1 mg/mL, though some applications may require different concentrations such as 100 μg/mL depending on your specific experimental needs. Avoid reconstituting to concentrations below 100 μg/mL, as this is not recommended for optimal protein stability.

Mixing and Dissolution

Mix the protein gently by swirling or inverting the vial—do not use vortexing or pipetting, as these methods can cause protein denaturation. Allow the protein to fully dissolve at room temperature for 10–15 minutes.

Storage and Stability

Long-term Storage

Store lyophilized powder at -20°C to -80°C for up to 12 months. Use a manual defrost freezer and avoid repeated freeze-thaw cycles, which can compromise protein integrity.

Short-term Storage After Reconstitution

Reconstituted protein solution can be stored at 4–7°C for 2–7 days. For longer storage, aliquots of reconstituted samples remain stable at -20°C or below for up to 3 months.

Minimizing Freeze-Thaw Cycles

Aliquot the reconstituted solution into smaller portions immediately after preparation to minimize freeze-thaw cycles, which can reduce protein activity and stability.

Sample Preparation for Cell Culture Applications

Cell Culture Supernatants

Centrifuge cell culture media at 1,500–2,000 × g for 10 minutes at 4°C to remove cellular debris. Collect the supernatant and use immediately or store at -20°C or below, avoiding repeated freeze-thaw cycles.

Biological Activity Verification

The biological activity of reconstituted VCAM-1 can be verified through cell adhesion assays. When immobilized at 10 μg/mL with 100 μL/well, approximately 85–95% of U937 human histiocytic lymphoma cells will adhere after 1 hour incubation at room temperature.

Human VCAM-1 promotes adhesion of lymphocytes, monocytes, eosinophils, and basophils through binding to leukocyte integrins VLA-4 (alpha 4 beta 1) and alpha 4 beta 7.