Recombinant Human BCAM

Using Recombinant Human BCAM in research applications is valuable for studying cell adhesion, migration, differentiation, and disease mechanisms, particularly in cancer, hematopoiesis, and vascular biology.

Key scientific applications and rationale:

• Cell Adhesion and Migration Studies: BCAM (Basal Cell Adhesion Molecule, also known as CD239 or Lutheran antigen) is a laminin receptor that mediates cell adhesion to laminin isoforms, especially those containing the α5 chain, which are abundant in basement membranes. Recombinant BCAM can be used to model and quantify cell adhesion, migration, and transmigration processes in vitro, such as in cancer metastasis or stem cell homing.

• Cancer Research: BCAM is upregulated in various tumors (carcinomas, sarcomas, astrocytomas, melanomas) and is implicated in tumor cell adhesion, migration, and metastasis. Recombinant BCAM enables mechanistic studies of tumor cell interactions with the extracellular matrix and the identification of potential therapeutic targets or inhibitors.

• Hematopoiesis and Stem Cell Niche: BCAM is expressed on hematopoietic stem and progenitor cells (HSPCs), endothelial cells, and mesenchymal stromal cells, playing a role in stem cell migration, differentiation, and niche interactions. Recombinant BCAM can be used to dissect these processes and to develop or test reagents (e.g., antibodies, inhibitors) that modulate stem cell behavior.

• Vascular and Red Blood Cell Disorders: BCAM is involved in the adhesion of sickle red blood cells to the vascular wall, contributing to vaso-occlusive crises in sickle cell disease. Recombinant BCAM is useful for studying these adhesive interactions and for screening potential therapeutic agents that block BCAM-laminin binding.

• Functional Assays and Reagent Development: Recombinant BCAM serves as a standard or ligand in ELISA, flow cytometry, or cell-based assays to characterize BCAM-binding antibodies, inhibitors, or other interacting proteins. It is also used to validate antibody specificity and function.

Advantages of using recombinant protein:

• Consistency and Reproducibility: Recombinant BCAM provides a defined, consistent reagent, reducing variability compared to native protein preparations.
• Engineering Flexibility: Recombinant forms can be tagged (e.g., His-tag, Fc-fusion) for purification, detection, or functional studies.
• Ethical and Practical Benefits: Recombinant production avoids the need for animal or human tissue sources and allows for scalable, controlled manufacturing.

Summary of research contexts where recombinant human BCAM is especially useful:

• Mechanistic studies of cell-matrix interactions and signaling.
• Modeling tumor cell invasion and metastasis.
• Investigating stem cell migration and differentiation.
• Screening and validating therapeutic antibodies or small molecule inhibitors targeting BCAM.
• Developing diagnostic assays for diseases involving BCAM dysregulation.

In summary, Recombinant Human BCAM is a versatile tool for dissecting the molecular and cellular roles of BCAM in health and disease, enabling both basic research and translational applications.

You can use recombinant human BCAM as a standard for quantification or calibration in your ELISA assays, provided it is of high purity and its concentration is accurately determined. This is a common and accepted practice in quantitative ELISA, as most commercial BCAM ELISA kits use recombinant human BCAM as their standard.

Key considerations and best practices:

• Purity and Formulation: The recombinant BCAM should be highly purified. Carrier-free formulations are preferred if BSA or other carriers might interfere with your assay, but BSA-containing formulations can be used if compatible with your system.
• Concentration Determination: Ensure the recombinant BCAM standard is accurately quantified, ideally by a reliable method such as absorbance at 280 nm with a known extinction coefficient, or by amino acid analysis.
• Standard Curve Preparation: Prepare a serial dilution of the recombinant BCAM in the same buffer or matrix as your samples to minimize matrix effects and ensure accurate quantification.
• Validation: Confirm that your ELISA detects the recombinant BCAM with similar efficiency as the endogenous/native protein. This is especially important if your assay uses antibodies that might have different affinities for recombinant versus native forms.
• Storage: Follow best practices for storage (typically -20°C or -80°C for lyophilized or aliquoted protein) to maintain stability and activity.

Summary Table: Use of Recombinant BCAM as ELISA Standard

Conclusion:
Using recombinant human BCAM as a standard is scientifically valid and widely practiced for ELISA quantification, as long as you follow best practices for protein handling, quantification, and assay validation.

Recombinant Human BCAM has been validated for several applications in published research, primarily in the context of cancer biology, cell adhesion, migration, and as a tool for antibody development and functional assays.

Key validated applications include:

• Cell Adhesion Assays: Recombinant Human BCAM supports the adhesion of TE-85 human osteogenic sarcoma cells, with a defined ED₅₀ for this effect, demonstrating its functional activity in vitro.
• ELISA (Enzyme-Linked Immunosorbent Assay): Recombinant BCAM has been used as a standard and binding target in ELISA to quantify soluble BCAM in cell culture supernatants and to measure antibody affinity, such as in studies identifying BCAM as a therapeutic target in ovarian cancer.
• Antibody Binding and Screening: Recombinant BCAM is used to confirm the specificity and binding affinity of candidate antibodies, including in the development of antibody–drug conjugates (ADCs) and targeted therapies for cancers expressing BCAM.
• Functional Cell Migration and Spheroid Assays: Soluble recombinant BCAM has been used to study its effects on tumor cell migration, spheroid dispersion, and invasion in three-dimensional culture systems and ex vivo tissue models, particularly in ovarian cancer metastasis research.
• Quantitative Immunofluorescence (QIF): While not directly using recombinant protein as a probe, studies have used antibodies validated against recombinant BCAM to quantify its expression in tissue samples from various cancers, supporting its role as a biomarker and therapeutic target.
• Colony Formation and Differentiation Assays: Antibodies raised against recombinant BCAM have been used to investigate its role in hematopoietic stem/progenitor cell differentiation and migration, indicating its functional relevance in stem cell biology.

Additional technical applications, as inferred from product datasheets and research protocols, include:

• SDS-PAGE and Western Blotting: Recombinant BCAM is used as a molecular weight standard and for antibody validation.
• Bioactivity Assays: Functional studies to assess BCAM’s role in cell adhesion, migration, and interaction with laminin isoforms.

Summary Table: Validated Applications for Recombinant Human BCAM

These applications demonstrate that recombinant human BCAM is a versatile tool in cancer research, cell biology, and therapeutic antibody development, with robust validation in both functional and analytical assays.

To reconstitute and prepare Recombinant Human BCAM protein for cell culture experiments, follow these steps to ensure protein integrity and biological activity:

1. Preparation Before Reconstitution

   • Store the lyophilized BCAM protein at -20°C to -80°C until use.
   • Before opening, centrifuge the vial briefly to collect all powder at the bottom and minimize loss.

2. Reconstitution

   • Add sterile distilled water or the buffer recommended in the product datasheet to the vial. Common reconstitution concentrations are 0.1–0.5 mg/mL.
   • Gently pipette or swirl to dissolve. Do not vortex or shake vigorously, as this can denature the protein and reduce activity.
   • Allow the solution to sit at room temperature for 15–30 minutes to ensure complete dissolution.

3. Dilution for Cell Culture

   • Dilute the reconstituted protein to your desired working concentration using cell culture medium or buffer containing a carrier protein (e.g., 0.1% BSA, 5% HSA, or 10% FBS).
   • Avoid using pure water for dilution, as this can cause protein degradation or loss due to adsorption to tube walls.
   • For serum-free or animal experiments, use trehalose as a stabilizer instead of animal-derived carriers.

4. Aliquoting and Storage

   • Use the reconstituted solution immediately for experiments, or aliquot and store at -20°C to -80°C for long-term storage.
   • Avoid repeated freeze-thaw cycles to preserve protein activity.
   • For short-term use (2–7 days), store at 4–8°C.
   • For long-term storage, consider adding 5–50% glycerol as a cryoprotectant.

5. Application in Cell Culture

   • Add the required amount of BCAM protein directly to the cell culture medium.
   • Change the medium regularly (every 2–3 days) as per standard cell culture protocols.

Key technical notes:

• Always consult the specific product datasheet or Certificate of Analysis for recommended buffer, concentration, and any special instructions.
• Use sterile technique throughout to prevent contamination.
• If the protein is tagged (e.g., His-tag), ensure compatibility with your downstream assay or cell type.

Summary Table: Recombinant Human BCAM Protein Reconstitution

Following these steps will help maintain the biological activity and stability of recombinant BCAM protein for cell culture experiments.