Recombinant Mouse CTLA-4 (CD152)

Recombinant Mouse CTLA-4 (CD152) is a valuable tool for immunological research, particularly in studies involving mouse models of immune regulation, autoimmunity, transplantation, and cancer immunotherapy. Here are several key reasons why you should consider using Recombinant Mouse CTLA-4 (CD152) in your research applications:

1. Functional Immune Modulation

• Recombinant Mouse CTLA-4 acts as a negative regulator of T cell activation by binding to B7-1 (CD80) and B7-2 (CD86) on antigen-presenting cells (APCs), thereby inhibiting IL-2 production and arresting T cells in the G1 phase of the cell cycle.
• This enables researchers to study the mechanisms of immune tolerance, T cell anergy, and the regulation of immune responses in vitro and in vivo.

2. High Purity and Consistency

• Commercially available recombinant proteins (e.g., from R&D Systems, BioLegend, Leinco, Acro Biosystems) are typically >97% pure and produced under controlled conditions, ensuring reproducibility and reliability in experimental results.

3. Versatile Applications

• Bioassays: Used to assess the functional impact of CTLA-4 on T cell activation, cytokine production, and immune cell interactions.
• Flow Cytometry: Can be used as a blocking agent or to study receptor-ligand interactions.
• Neutralization Studies: Allows for the investigation of CTLA-4-mediated immune suppression and its reversal in experimental models.
• Preclinical Therapeutic Evaluation: Useful for testing the effects of CTLA-4 blockade or agonism in mouse models of disease.

4. Species-Specific Relevance

• Mouse CTLA-4 is essential for studies using murine models, which are widely used in immunology due to their genetic similarity to humans and the availability of well-characterized knockout and transgenic strains.
• Using the mouse-specific protein ensures accurate and physiologically relevant results in mouse-based experiments.

5. Support for Mechanistic Studies

• Enables detailed investigation of CTLA-4 signaling pathways, including its role in dendritic cell maturation, thymocyte development, and the induction of immunological tolerance.
• Facilitates the study of CTLA-4's involvement in autoimmune diseases, transplant rejection, and tumor immunology.

6. Compatibility with Other Research Tools

• Recombinant Mouse CTLA-4 can be used in conjunction with anti-CTLA-4 antibodies (e.g., clones 9D9, 9H10, UC10-4F10-11) for neutralization or blocking experiments, allowing for comprehensive analysis of CTLA-4 function.

7. Standardization and Reproducibility

• The use of recombinant protein ensures a standardized reagent across experiments, reducing variability and enhancing the comparability of results between different studies and laboratories.

In summary, Recombinant Mouse CTLA-4 (CD152) is an essential reagent for researchers studying immune regulation, tolerance, and checkpoint inhibition in mouse models. Its high purity, functional activity, and compatibility with a wide range of applications make it a reliable choice for advancing immunological research.

Recombinant Mouse CTLA-4 (CD152) protein can be used as a standard for quantification or calibration in ELISA assays, provided it is validated for this purpose and matches the assay’s requirements. Recombinant standards are commonly used to generate standard curves for quantifying target proteins in ELISA, allowing for accurate measurement of CTLA-4 concentrations in biological samples.

Essential context and best practices:

• Validation: Ensure the recombinant CTLA-4 protein is compatible with your ELISA system. Most commercial ELISA kits for mouse CTLA-4 include a recombinant protein standard specifically formulated and validated for use in their assay protocols. If using a recombinant protein from a different source, confirm its purity, concentration, and biological activity, and verify that it is recognized by the capture and detection antibodies in your assay.

• Standard Curve Preparation: Prepare serial dilutions of the recombinant CTLA-4 protein to generate a standard curve covering the expected concentration range of your samples. The standard curve allows interpolation of sample concentrations based on their absorbance or signal values.

• Protein Format: Recombinant CTLA-4 proteins may be supplied as Fc chimeras, His-tagged, or untagged forms. The format should not interfere with antibody recognition in your ELISA. For quantification, the protein should be well-characterized, with known concentration and purity (typically >95%).

• Assay Compatibility: Some recombinant CTLA-4 proteins are labeled for use in bioassays, while others are specifically recommended for ELISA calibration. Proteins not validated for ELISA may yield inaccurate results due to differences in folding, glycosylation, or epitope presentation.

• Storage and Handling: Follow manufacturer recommendations for storage and handling to maintain protein integrity. Avoid repeated freeze-thaw cycles and use freshly prepared standards for each assay to ensure consistency.

Additional relevant information:

• Matched Antibody Pairs: If developing a custom ELISA, use matched antibody pairs validated for mouse CTLA-4 detection, and confirm that your recombinant standard is recognized by these antibodies.

• Documentation: Refer to the datasheet or technical documentation for your recombinant CTLA-4 protein to confirm its intended use as an ELISA standard and any specific instructions regarding dilution, stability, and compatibility.

In summary, recombinant mouse CTLA-4 can be used as a standard for ELISA quantification if it is validated for this application and matches the assay’s antibody specificity and detection format. Always verify compatibility and follow best practices for standard curve preparation and protein handling.

Recombinant Mouse CTLA-4 (CD152) has been validated for several key applications in published research, primarily in immunological and cell biology studies.

The main validated applications include:

• Bioassays: Used to study CTLA-4-mediated inhibition of T cell activation, such as suppression of IL-2 secretion in stimulated Jurkat cells and inhibition of IFN-γ production by NK cells. Bioassays have also been used to investigate CTLA-4’s role in dendritic cell maturation, T cell anergy, and immune tolerance in various mouse models.

• Neutralization: Applied to block CTLA-4 function in vitro and in vivo, enabling studies of immune checkpoint blockade and its effects on T cell co-stimulation and anti-tumor immunity.

• Flow Cytometry: Used for detection and quantification of CTLA-4 expression on immune cell subsets, including T cells and NK cells.

• Western Blot (WB): Validated for detection of CTLA-4 protein in mouse splenocytes and other tissues.

• Immunohistochemistry (IHC) and Immunofluorescence (IF): Used to localize and quantify CTLA-4-positive lymphocytes in tissue sections, particularly in lymphoid organs.

• ELISA: Applied for quantification of CTLA-4 in biological samples.

Supporting details and context:

• Functional studies: Recombinant Mouse CTLA-4 has been used to inhibit T cell activation by reducing IL-2 production and IL-2 receptor expression, and to arrest T cells in the G1 phase of the cell cycle. It has also been used to study interactions with antigen-presenting cells (APCs), B cells, and ligands such as CD80 (B7.1) and CD86 (B7.2).

• Preclinical models: The protein has been employed in mouse models to investigate immune regulation, tumor immunity, and the effects of CTLA-4 blockade in cancer and transplantation research.

• Combination therapies: Studies have validated its use in combination with other immune checkpoint inhibitors (e.g., anti-PD-1 antibodies) to assess synergistic effects on tumor growth inhibition and immune cell infiltration in mouse models.

• Tolerance and autoimmunity: Recombinant CTLA-4 has been used to explore mechanisms of immunological tolerance, thymocyte development, and prevention of autoimmunity.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and product validation data, confirming the utility of recombinant mouse CTLA-4 (CD152) in diverse immunological research contexts.

To reconstitute and prepare Recombinant Mouse CTLA-4 (CD152) protein for cell culture experiments, follow these general steps, which are consistent with best practices for recombinant protein handling and are supported by multiple technical datasheets:

1. Centrifuge the vial before opening:
This ensures all lyophilized protein is collected at the bottom of the vial and prevents loss during opening.

2. Choose an appropriate buffer:

• Sterile PBS (phosphate-buffered saline), pH 7.2–7.4, is commonly recommended for reconstitution.
• If the protein is supplied with carrier protein (e.g., BSA), reconstitute in PBS containing at least 0.1%–1% BSA or HSA to stabilize the protein and prevent adsorption to plastic.
• For carrier-free proteins, you may add 0.1%–1% BSA if your downstream application allows, especially for low-concentration working stocks.

3. Reconstitution concentration:

• A typical reconstitution concentration is 200 μg/mL in sterile PBS (with or without carrier protein, depending on your application and the supplied formulation).
• Some protocols recommend not reconstituting below 100 μg/mL to maintain protein stability.

4. Gentle mixing:

• Gently swirl or invert the vial to dissolve the protein. Do not vortex or pipette up and down vigorously, as this can denature the protein.
• Allow the protein to fully dissolve at room temperature for 10–30 minutes.

5. Aliquot and storage:

• Once reconstituted, aliquot the protein to avoid repeated freeze-thaw cycles, which can degrade activity.
• Store aliquots at –20°C or –70°C for long-term storage. For short-term use (up to 1 week), store at 2–8°C.
• Avoid repeated freeze/thaw cycles.

6. Working solution preparation:

• For cell culture experiments, dilute the reconstituted stock to the desired working concentration using sterile PBS or cell culture medium, ideally containing 0.1%–1% BSA or HSA if compatible with your assay.
• Filter-sterilize the working solution if sterility is critical and the protein is not already supplied sterile.

Summary Table: Recombinant Mouse CTLA-4 (CD152) Protein Reconstitution

Additional notes:

• Always consult the specific Certificate of Analysis (CoA) or datasheet for your protein lot, as formulations and recommended procedures may vary slightly between suppliers.
• If using for functional cell-based assays, ensure the protein is endotoxin-free or has endotoxin levels suitable for cell culture (<1 EU/mg is typical).
• For ELISA or plate-coating applications, reconstitution in PBS alone is usually sufficient.

These guidelines will help ensure optimal solubility, stability, and biological activity of recombinant mouse CTLA-4 (CD152) protein for cell culture experiments.