Activated leukocyte cell adhesion molecule (ALCAM/CD166) is an 82 kDa transmembrane glycoprotein and a member of the immunoglobulin gene superfamily. It is also known as MEMD, SC-1/DM-GRASP and KG-CAM. It is linked with cell migration and development and with cancer progression (1). ALCAM is expressed on natural killer cells, thymocytes, platelets, B cells, T cells and monocytes after cell activation, as well as eosinophils, fibroblasts, endothelial cells, keratinocytes and neurons (2). It is a ligand for CD6 and NgCAM but also engages in homophilic interactions.
ALCAM/CD6 interaction plays a role in T cell development and T cell regulation (3).
Protein Details
Purity
>95% by SDS-PAGE and analyzed by silver stain.
Endotoxin Level
<0.1 EU/µg as determined by the LAL method
Biological Activity
The biological activity of Mouse ALCAM was determined by its ability to block adhesion of HUT-78, a human cutaneous T cell lymphoma, to immobilized rhCD6/Fc protein. rmALCAM/Fc at 10 μg/mL (50 μL/well) blocked 100% of HUT-78 cell adhesion (1 x 106 cells/mL, 50 μL/well) to immobilized rhCD6/Fc (10 μg/mL, 100 μL/well).
The predicted molecular weight of Recombinant Mouse ALCAM is Mr 82 kDa. However, the actual molecular weight as observed by migration on SDS-PAGE is Mr 114-122 kDa.
Predicted Molecular Mass
82
Formulation
This recombinant protein was 0.2 µm filtered and lyophilized from modified Dulbecco’s phosphate buffered saline (1X PBS) pH 7.2 – 7.3 with no calcium, magnesium, or preservatives.
Storage and Stability
This lyophilized protein is stable for six to twelve months when stored desiccated at -20°C to -70°C. After aseptic reconstitution, this protein may be stored at 2°C to 8°C for one month or at -20°C to -70°C in a manual defrost freezer. Avoid Repeated Freeze Thaw Cycles. See Product Insert for exact lot specific storage instructions.
Powered by AI: AI is experimental and still learning how to provide the best assistance. It may occasionally generate incorrect or incomplete responses. Please do not rely solely on its recommendations when making purchasing decisions or designing experiments.
Recombinant Mouse ALCAM (Activated Leukocyte Cell Adhesion Molecule) is a valuable tool for research applications due to its well-characterized biological functions and the advantages conferred by recombinant protein technology. Here are several reasons why you should consider using Recombinant Mouse ALCAM in your research:
1. Biological Relevance and Functional Studies
Cell Adhesion and Migration: ALCAM is a key cell adhesion molecule involved in leukocyte transmigration, dendritic cell migration, and maintenance of blood-brain barrier (BBB) integrity. Using recombinant ALCAM allows you to study these processes in vitro and in vivo, including its role in neuroinflammation and immune responses.
BBB Integrity: Studies have shown that ALCAM knockout mice exhibit increased BBB permeability and more severe experimental autoimmune encephalomyelitis (EAE), highlighting ALCAM's role in maintaining tight junction stability and BBB integrity. Recombinant ALCAM can be used to investigate these mechanisms and their implications in neurological diseases.
Apoptosis Regulation: Recombinant ALCAM has been shown to reduce cell death progression in TGF-β1–treated cells, indicating its protective role in apoptosis. This makes it useful for studying cell survival and death pathways.
2. Consistency and Reproducibility
High Purity and Consistency: Recombinant proteins are produced under controlled conditions, ensuring high purity and batch-to-batch consistency. This is crucial for reproducible experimental results.
Defined Structure: The recombinant form provides a defined and consistent protein structure, which is essential for functional assays and structural studies.
3. Versatility in Assays
Biological Assays: Recombinant Mouse ALCAM can be used in a variety of biological assays, including cell adhesion, migration, and signaling studies. It can also be used to assess the effects of ALCAM on cell survival and death.
Structural Studies: The recombinant protein can be used for structural biology studies, such as X-ray crystallography and electron cryomicroscopy, to determine the three-dimensional structure of ALCAM and its complexes.
Therapeutic Development: Recombinant ALCAM can be used to develop and test therapeutic strategies, such as blocking ALCAM to modulate immune responses or to study its role in disease progression.
4. Ease of Use and Customization
Tagged Forms: Many recombinant ALCAM proteins are available with tags (e.g., His-tag, Fc-tag) that facilitate purification, detection, and conjugation. These tags can be used for immunoprecipitation, Western blotting, and other downstream applications.
Customization: Recombinant technology allows for the engineering of ALCAM with additional functionalities, such as conjugation sites for labeling or mutagenesis to study specific domains or interactions.
5. Research Applications
Neuroscience: Study the role of ALCAM in neuroinflammation, BBB integrity, and neurodegenerative diseases.
Immunology: Investigate ALCAM's role in immune cell migration, dendritic cell function, and immune responses.
Cancer Biology: Explore ALCAM's involvement in cancer cell invasion, tumor angiogenesis, and chemoresistance.
Cell Biology: Use ALCAM to study cell adhesion, migration, and signaling pathways.
6. Availability and Reliability
Commercial Availability: Recombinant Mouse ALCAM is available from multiple suppliers, ensuring reliable and consistent access to the protein for your research needs.
Research Use Only: These proteins are provided for research use only, making them suitable for a wide range of experimental applications.
In summary, Recombinant Mouse ALCAM is a powerful and versatile tool for studying cell adhesion, migration, BBB integrity, and various disease processes. Its high purity, consistency, and ease of use make it an excellent choice for a wide range of research applications.
You can use recombinant Mouse ALCAM as a standard for quantification or calibration in your ELISA assays, provided it is properly characterized and matches the form of ALCAM detected by your assay. Recombinant proteins are commonly used as standards in ELISA, especially when purified native protein is unavailable.
Key considerations for using recombinant Mouse ALCAM as an ELISA standard:
Protein Form: The standard should match the form of ALCAM detected by your assay (e.g., extracellular domain, full-length, or specific isoform). Most commercial ELISA kits use the mature extracellular domain as the standard.
Purity and Quantification: The recombinant protein must be highly purified and accurately quantified, ideally by methods such as HPLC or absorbance at 280 nm with a known extinction coefficient.
Standard Curve Preparation: Prepare a fresh standard curve for each assay using serial dilutions of the recombinant protein in the same buffer or matrix as your samples to minimize matrix effects.
Validation: Confirm that the recombinant standard produces a parallel dose-response curve to native ALCAM in your sample matrix, ensuring accurate quantification. This is especially important if your samples are complex (e.g., tissue lysates or serum).
Carrier Proteins: If your recombinant ALCAM is supplied carrier-free, consider adding a carrier protein (e.g., BSA) to prevent adsorption losses during dilution and storage.
Limitations and best practices:
Kit Compatibility: If you are using a commercial ELISA kit, check the kit documentation to ensure compatibility with external standards. Some kits are optimized for their included standards and may not guarantee performance with others.
Standard Stability: Prepare standards fresh for each assay, as working dilutions of recombinant proteins may not be stable over time.
Documentation: Record the lot number, concentration, and preparation details of your recombinant standard for reproducibility.
In summary: Recombinant Mouse ALCAM is suitable as a standard for ELISA quantification if it is well-characterized, matches the assay’s target, and is validated for parallelism with native samples. Always follow best practices for standard preparation and validation to ensure accurate and reliable quantification.
Recombinant Mouse ALCAM (Activated Leukocyte Cell Adhesion Molecule, CD166) has been validated in published research for several key applications, primarily in functional assays, cell adhesion studies, ELISA, and as a tool for blocking or neutralizing ALCAM-mediated interactions.
Key validated applications include:
Cell adhesion and migration assays: Recombinant Mouse ALCAM has been used to study homotypic (ALCAM–ALCAM) and heterotypic (ALCAM–CD6) cell adhesion, as well as leukocyte transmigration and dendritic cell migration across endothelial barriers. These assays help elucidate ALCAM’s role in immune cell trafficking, angiogenesis, and tissue inflammation.
Blocking/neutralization studies: The recombinant protein is used as a competitor or blocking agent to disrupt ALCAM-mediated interactions, such as in competition ELISA to block ALCAM–CD6 binding, or in functional assays to inhibit leukocyte or dendritic cell transmigration. This is critical for validating ALCAM as a therapeutic target in models of inflammation, transplantation, and cancer metastasis.
ELISA (Enzyme-Linked Immunosorbent Assay): Recombinant Mouse ALCAM is commonly used as a standard or as a capture antigen in ELISA to quantify ALCAM or to assess the binding of antibodies or ligands to ALCAM.
Protein-protein interaction studies: The recombinant protein is used to characterize binding partners (e.g., CD6) and to map interaction domains, often employing Fc-chimera or His-tagged formats for immobilization and detection.
In vitro and in vivo functional validation: Recombinant Mouse ALCAM has been used in mouse models to validate its role in processes such as T cell activation, angiogenesis, lymphangiogenesis, and immune cell trafficking, often by administering blocking antibodies or recombinant fragments that target ALCAM.
Cancer research: It is used to study the role of ALCAM in tumor cell adhesion, migration, and metastasis, particularly in gastrointestinal and pancreatic cancer models, where ALCAM expression correlates with metastatic potential.
Summary Table of Validated Applications
Application Type
Description/Context
Cell adhesion/migration assays
Leukocyte, dendritic cell, and tumor cell adhesion and transmigration studies
Blocking/neutralization
Functional inhibition of ALCAM–CD6 and ALCAM–ALCAM interactions
ELISA
Quantification and ligand/antibody binding studies
Protein-protein interaction
Mapping and validation of ALCAM binding partners
In vivo functional studies
Validation in mouse models of inflammation, transplantation, and cancer
Additional notes:
Recombinant Mouse ALCAM is typically expressed in HEK293 cells and is available in various tagged formats (e.g., His-tag, Fc-chimera) to facilitate different assay requirements.
It is not typically used for flow cytometry or Western blot as a primary reagent, but rather as a functional or blocking protein in cell-based and biochemical assays.
If you require protocol-specific details or references for a particular application, please specify the assay or research context.
To reconstitute and prepare Recombinant Mouse ALCAM protein for cell culture experiments, dissolve the lyophilized protein in sterile, endotoxin-free water or an appropriate buffer (such as PBS or Tris-buffered saline), following the concentration recommended on the product datasheet or vial label. Mix gently and allow the protein to fully dissolve at room temperature for 15–30 minutes, using gentle agitation; if visible particulates remain, extend mixing up to 2 hours.
Essential steps and considerations:
Equilibration: Bring both the lyophilized protein vial and the chosen buffer to room temperature before reconstitution to avoid condensation and ensure proper dissolution.
Centrifugation: Briefly centrifuge the vial to collect all powder at the bottom before adding buffer.
Buffer selection: Use sterile, endotoxin-free water for most applications, unless the datasheet specifies a different buffer (e.g., PBS, Tris-HCl). For cell culture, ensure the buffer is compatible with your downstream assay and does not contain toxic additives.
Concentration: Prepare a stock solution at the recommended concentration (e.g., 0.5 mg/mL). Adjust as needed for your experimental design.
Mixing: Gently mix by inversion or pipetting. Avoid vigorous vortexing, which may denature the protein.
Incubation: Allow the protein to dissolve for 15–30 minutes at room temperature. If undissolved material remains, continue gentle mixing for up to 2 hours.
Sterile filtration: For cell culture use, filter the reconstituted protein through a 0.22 μm sterile filter to remove particulates and ensure sterility.
Aliquoting and storage: Aliquot the stock solution to avoid repeated freeze-thaw cycles. Store at −80°C for long-term storage; avoid storing at 37°C or repeated freeze-thawing, which can degrade the protein.
Carrier protein: If recommended, add a carrier protein such as BSA (0.1–1 mg/mL) to enhance stability, especially at low concentrations.
Endotoxin testing: Confirm endotoxin levels are suitable for cell culture (<0.1 EU/μg is typical for sensitive applications).
Preparation for cell culture experiments:
Dilution: Dilute the stock solution into cell culture medium immediately before use, ensuring the final concentration matches your experimental requirements.
Compatibility: Confirm that the buffer used for reconstitution is compatible with your cell culture system and does not affect cell viability or function.
Summary Table: Recombinant Mouse ALCAM Protein Reconstitution
Step
Details
Equilibration
Room temperature for vial and buffer
Centrifugation
Brief spin to collect powder
Buffer
Sterile water or PBS (check datasheet)
Concentration
As recommended (e.g., 0.5 mg/mL)
Mixing
Gentle inversion/pipetting, 15–30 min (up to 2 h if needed)
Filtration
0.22 μm sterile filter for cell culture
Aliquoting/Storage
Aliquot, store at −80°C, avoid freeze-thaw cycles
Carrier Protein
Optional BSA addition for stability
Endotoxin
<0.1 EU/μg for cell culture
Dilution
Dilute in cell culture medium before use
Always consult the specific product datasheet for any unique instructions regarding buffer composition, concentration, or storage conditions.
References & Citations
1. Weichert, W. et al. (2004) J. Clin. Pathol. 57:1160
2. Bowen, MA. et al. (1995) J. Exp. Med. 181:2213
3. Aruffo, A. et al. (1997) Immunol. Today 18:498
Products are for research use only. Not for use in diagnostic or therapeutic procedures.
