Recombinant Mouse CD6

Recombinant Mouse CD6 is a valuable tool in research applications focused on immunology, autoimmunity, and therapeutic antibody development because it enables precise study and manipulation of CD6-mediated T cell responses, antibody generation, and disease modeling.

Key scientific applications and advantages:

• Antibody Development: Recombinant mouse CD6 is essential for generating monoclonal antibodies against CD6. For example, immunizing CD6 knockout mice with recombinant mouse CD6 protein allows for the production of mouse anti-mouse CD6 monoclonal antibodies, which can be screened for cross-reactivity with human CD6 and used in therapeutic studies.

• Functional Studies: Recombinant CD6 enables in vitro assays to dissect the role of CD6 in T cell activation, proliferation, and differentiation. CD6 is a costimulatory molecule that regulates T cell responses via interaction with its ligand ALCAM, making it central to studies of immune synapse formation and signaling.

• Autoimmunity and Disease Modeling: CD6 is implicated in several autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE, a model for multiple sclerosis), autoimmune uveitis, and rheumatoid arthritis. Recombinant CD6 can be used to study disease mechanisms, test therapeutic antibodies, and develop new immunomodulatory strategies.

• Therapeutic Screening: Recombinant mouse CD6 is used to screen and characterize antibodies or small molecules targeting CD6, facilitating preclinical evaluation of potential immunosuppressive or immunomodulatory agents.

• Cellular Interaction Studies: CD6 is involved in cell-to-cell contact and immune synapse formation. Recombinant CD6 protein allows for biochemical and cell-based assays to study CD6-ALCAM interactions and downstream signaling pathways.

Best practices for use:

• Use recombinant mouse CD6 in ELISA, flow cytometry, and immunization protocols to generate and validate anti-CD6 antibodies.
• Employ recombinant CD6 in functional assays to assess T cell activation, cytokine production, and proliferation in response to CD6 engagement.
• Integrate recombinant CD6 in disease models to evaluate the efficacy of CD6-targeted therapies and understand CD6’s role in pathogenesis.

Summary:
Recombinant Mouse CD6 is indispensable for antibody development, mechanistic immunology studies, and preclinical therapeutic screening, especially in the context of T cell-mediated autoimmune diseases and immune regulation.

You can use recombinant Mouse CD6 as a standard for quantification or calibration in your ELISA assays, provided it is of high purity and its concentration is accurately known. This is a common and accepted practice in ELISA development and quantification.

Key considerations and best practices:

• Purity and Characterization: The recombinant CD6 protein should be well-characterized and as pure as possible, since impurities can affect the accuracy of your standard curve.
• Standard Curve Preparation: Prepare a serial dilution of the recombinant CD6 in the same buffer or diluent used for your samples to generate a standard curve covering the expected concentration range. Follow the ELISA kit or assay protocol for recommended dilution ranges and procedures.
• Immunoreactivity: Ensure that the recombinant CD6 is recognized by the capture and detection antibodies used in your ELISA. Most commercial ELISA kits for mouse CD6 are validated to detect both natural and recombinant forms.
• Lot-to-Lot Variation: There may be slight differences in immunoreactivity or apparent concentration between different lots of recombinant protein. It is best to assign the standard curve values based on the ELISA readout rather than relying solely on the mass stated on the vial.
• Controls: Include appropriate controls, such as endogenous positive controls (e.g., mouse serum with known CD6 levels), to validate the performance of your standard and the assay as a whole.
• Reconstitution and Storage: If your recombinant CD6 is lyophilized, reconstitute it according to the manufacturer’s instructions and avoid repeated freeze-thaw cycles, as this can affect protein integrity and quantification accuracy.

Limitations:

• If your recombinant CD6 is not specifically validated for use as an ELISA standard, you should verify its performance by comparing it to a reference standard or by spiking experiments.
• The standard curve should be freshly prepared for each assay, as protein degradation or adsorption to tubes can alter concentrations over time.

Summary Table:

In summary, recombinant Mouse CD6 is suitable as a standard for ELISA quantification if you follow best practices for preparation, validation, and use.

Recombinant Mouse CD6 has been validated for several key applications in published research, primarily in immunology and cell biology:

• Generation of monoclonal antibodies: Recombinant mouse CD6 has been used as an immunogen to produce mouse anti-mouse CD6 monoclonal antibodies. These antibodies are then screened for specificity and cross-reactivity using ELISA and flow cytometry assays.

• Flow cytometry: Recombinant CD6 is used to screen and validate antibody binding to the extracellular regions of mouse CD6 on T cells under physiological conditions.

• ELISA (Enzyme-Linked Immunosorbent Assay): It serves as a target antigen for identifying hybridoma clones producing anti-CD6 antibodies.

• Cell adhesion assays: Immobilized recombinant mouse CD6 supports the adhesion of Jurkat cells, a human T cell line, demonstrating its functional role in cell-cell interactions.

• Functional studies in autoimmune disease models: Recombinant mouse CD6 has been used in vivo to study its role in experimental autoimmune uveitis (EAU) and multiple sclerosis (EAE) models, including immunization protocols and therapeutic antibody development.

• Removal of CD6+ T cells: Recombinant CD6 has been applied to selectively deplete CD6+ T cells from donor bone marrow prior to allogenic bone marrow transplantation.

• Antigen-specific recall assays: Used to assess the elimination of pathogenic T cells in vitro, particularly in studies involving antibody-drug conjugates targeting CD6.

Summary Table of Validated Applications

These applications demonstrate the versatility of recombinant mouse CD6 in both basic research and translational studies, particularly in the context of T cell biology, autoimmunity, and therapeutic antibody development.

To reconstitute and prepare Recombinant Mouse CD6 protein for cell culture experiments, dissolve the lyophilized protein in sterile buffer—typically 10 mM PBS (pH 7.4) or sterile distilled water—to a final concentration between 0.1–1.0 mg/mL. Avoid vortexing or vigorous pipetting to prevent protein denaturation.

Step-by-step protocol:

• Centrifuge the vial briefly before opening to ensure all powder is at the bottom.
• Add sterile buffer (e.g., 10 mM PBS, pH 7.4) or sterile distilled water. The recommended concentration is 0.1–1.0 mg/mL; for example, add 1 mL buffer to 1 mg protein for 1 mg/mL.
• Gently mix by inverting the tube or allowing the solution to sit at room temperature for 10 minutes. Do not vortex or pipette vigorously.
• Aliquot the solution to avoid repeated freeze-thaw cycles.
• Storage:
  • Short-term: 2–8 °C for up to one month.
  • Long-term: –20 °C to –80 °C for up to 12 months.
  • Avoid repeated freeze-thaw cycles to maintain protein integrity.

Preparation for cell culture experiments:

• Dilute the reconstituted stock to the desired working concentration using sterile cell culture medium or PBS, depending on your assay requirements.
• Filter-sterilize the final working solution if necessary (0.2 μm filter) to ensure sterility for cell culture applications.
• Carrier proteins (e.g., 0.1% BSA) may be added to working solutions to stabilize the protein, especially at low concentrations.

Additional notes:

• Do not reconstitute below 100 μg/mL unless specified by your protocol, as lower concentrations may reduce stability.
• Always consult the specific product datasheet for any unique requirements regarding buffer composition or additives.

Summary of best practices:

• Use gentle mixing.
• Avoid vortexing.
• Aliquot and freeze for long-term storage.
• Use sterile technique throughout.

These steps will ensure optimal solubility, stability, and biological activity of recombinant Mouse CD6 protein for cell culture experiments.