Recombinant Human ALCAM

Recombinant human ALCAM (Activated Leukocyte Cell Adhesion Molecule, also known as CD166) offers substantial value across multiple research applications due to its diverse biological roles and well-characterized functions in cell-cell interactions and disease pathology.

Cancer Research Applications

Recombinant ALCAM serves as a powerful tool for investigating cancer biology and developing therapeutic strategies. The protein is highly expressed on numerous cancer cell lines across various origins, making it an excellent target for cancer-specific research. ALCAM plays a critical role in cancer cell invasion and metastasis, with studies demonstrating that antibodies targeting ALCAM can inhibit breast cancer cell invasion by approximately 50% in in vitro invasion assays and reduce tumor growth in vivo models. Additionally, ALCAM expression correlates with histological tumor grade, positioning it as a valuable biomarker for cancer progression assessment. In multiple myeloma, ALCAM negatively regulates clonogenicity, and patients with high ALCAM expression demonstrate superior overall survival, suggesting its importance in understanding chemoresistance mechanisms. For prostate cancer research, ALCAM appears as a promising biomarker for disease progression, with enhanced serum expression associated with poorer prognosis.

Blood-Brain Barrier and Neuroinflammation Studies

Recombinant ALCAM is invaluable for investigating blood-brain barrier (BBB) integrity and neuroinflammatory processes. ALCAM maintains tight junction stability at the BBB by acting as an adaptor molecule that indirectly links junctional proteins to the cytoskeleton. High ALCAM expression at the blood-brain barrier in active multiple sclerosis and its animal models promotes leukocyte migration to the brain. Research using ALCAM knockout models has revealed that ALCAM regulates the expression of critical junctional proteins including occludin, ZO-1, and α-catenin, making recombinant ALCAM essential for understanding BBB permeability mechanisms.

Immunological and Cell Adhesion Research

The protein is fundamental for studying cell-cell interactions and immune cell trafficking. ALCAM is required for normal organization of the lymph vessel network and normal hematopoietic stem cell engraftment in the bone marrow. Long-term engagement between CD6 and ALCAM is essential for T-cell proliferation induced by dendritic cells, making recombinant ALCAM useful for immunological assays and cell interaction studies.

Biomarker Development

Recombinant ALCAM serves as a reference standard for developing diagnostic assays and biomarker validation studies. Soluble ALCAM has been identified as a potential biomarker for epithelial ovarian cancer, with increased levels in type II tumors, and its relevance has been demonstrated in cervical cancer patient sera. The protein's cancer-specific binding pattern, with minimal staining in normal human tissues except for weak staining in some normal breast samples, makes it particularly suitable for developing targeted diagnostic applications.

Functional Assays and Drug Development

Recombinant ALCAM-Fc chimera constructs are particularly useful for functional studies, as they can inhibit cell attachment and modulate cellular responsiveness to microenvironmental factors. These constructs enable researchers to investigate ALCAM's role in cell adhesion networks and test potential therapeutic interventions targeting ALCAM-mediated interactions.

You can use recombinant human ALCAM as a standard for quantification or calibration in your ELISA assays, provided it is of high purity, its concentration is accurately known, and it is compatible with the antibodies and detection system used in your assay.

Key considerations:

• Source and Compatibility: Many commercial ELISA kits use recombinant human ALCAM as the standard, and it is generally suitable for generating standard curves in sandwich ELISAs targeting human ALCAM. However, you must ensure that the recombinant protein is recognized by the capture and detection antibodies in your specific assay format.
• Concentration Calibration: The recombinant standard must have a precisely determined concentration. Any error in the concentration of your standard will directly affect the accuracy of your quantification.
• Matrix Effects: For best accuracy, dilute your recombinant standard in the same matrix as your samples (e.g., serum, plasma, or buffer) to minimize matrix effects and ensure comparable recovery.
• Validation: If you are not using the standard provided with a commercial kit, you should validate your recombinant ALCAM standard by running a spike-and-recovery experiment and comparing its performance to the kit standard, if available.
• Documentation: Some vendors note that their recombinant ALCAM protein is suitable for use as a standard in sandwich ELISA, but caution that concentration calibration may differ from kit standards, so results may not be directly comparable between different sources or lots.

Protocol tips:

• Prepare a serial dilution of the recombinant ALCAM to generate a standard curve covering the expected range of your samples.
• Use 4-parameter or 5-parameter logistic curve fitting for best accuracy in quantification.
• Always include a zero standard (blank) and run standards in duplicate or triplicate for reliability.

Summary:
Recombinant human ALCAM is widely used as a standard in ELISA assays for quantification, but you must ensure its compatibility with your assay system, validate its performance, and use proper calibration and dilution protocols for accurate results.

Validated Research Applications

Recombinant human ALCAM (activated leukocyte cell adhesion molecule, also known as CD166) has been validated across multiple research applications in published studies:

Cell Adhesion and Migration Studies

Recombinant ALCAM has been extensively used to investigate cell-cell adhesion mechanisms. The protein effectively blocks adhesion of T lymphocytes to immobilized CD6, with demonstrated potency (ED50 of 0.4-1.6 µg/mL). Additionally, recombinant ALCAM-Fc chimera constructs have been employed to study attachment inhibition in various cell types, including colorectal carcinoma and osteosarcoma-derived fibroblasts. The protein has also been validated for promoting CD6highCD4+ T cell migration in vitro using primary human CD4+ T cells isolated from peripheral blood mononuclear cells.

Neurodevelopmental Research

ALCAM has been validated for studying neurite extension and axon growth. The protein promotes axon guidance and mediates outgrowth and pathfinding for retinal ganglion cell axons, making it valuable for developmental neurobiology research.

Bioassay Development

Recombinant ALCAM serves as a standard for bioassays examining immunological interactions. It has been validated in functional assays measuring T cell proliferation induced by dendritic cells, where long-term engagement of CD6 and ALCAM was shown to be essential. The protein is also suitable for use as an ELISA standard in immunoassay applications.

Cancer Research Applications

The protein has been utilized in cancer cell biology research to investigate ALCAM's role in tumor progression, invasion, and metastasis across multiple cancer types including melanoma, breast cancer, colorectal carcinoma, and ovarian cancer.

To reconstitute and prepare Recombinant Human ALCAM protein for cell culture experiments, dissolve the lyophilized protein in sterile PBS at a concentration of 100 μg/mL. Use gentle agitation at room temperature for 15–30 minutes to ensure complete dissolution, and avoid vigorous shaking to prevent foaming and protein denaturation.

Detailed protocol and best practices:

• Reconstitution:

  • Briefly centrifuge the vial to collect the powder at the bottom before opening.
  • Add sterile PBS to achieve the desired concentration (commonly 100 μg/mL for ALCAM).
  • Gently mix by inverting or slow pipetting; do not vortex or shake vigorously.
  • Allow the solution to sit at room temperature for 15–30 minutes to fully dissolve.

• Aliquoting and Storage:

  • Once dissolved, aliquot the solution to avoid repeated freeze-thaw cycles, which can degrade the protein.
  • For long-term storage, keep aliquots at −20°C to −80°C.
  • For short-term use (2–7 days), store at 4–8°C.
  • Optionally, add a carrier protein (e.g., 0.1% BSA) or 5–50% glycerol to stabilize the protein during storage, especially for low-concentration working solutions.

• Preparation for Cell Culture:

  • Before use, thaw aliquots on ice and dilute to the working concentration required for your assay (e.g., 0.3–1.5 μg/mL for bioassays).
  • Filter the final solution through a 0.2 μm sterile filter if sterility is critical for cell culture applications.
  • Avoid exposing the protein to temperatures above room temperature and do not dissolve at 37°C, as this may cause denaturation.

• General Notes:

  • Always consult the specific product datasheet for recommended buffers and concentrations, as formulations may vary.
  • Optimal working concentrations should be determined empirically for each cell type and experimental setup.

Summary Table:

Following these steps will help maintain the integrity and biological activity of recombinant ALCAM for cell culture experiments.