Anti-Mouse CD28 Recombinant Protein

Applications and Benefits of Anti-Mouse CD28 Recombinant Protein

Anti-mouse CD28 recombinant proteins serve as essential tools for T cell immunology research due to their role in costimulatory signaling. CD28 is a critical costimulatory receptor that provides the second signal required for full T cell activation, working in conjunction with T cell receptor (TCR) signaling.

Key Research Applications

T Cell Activation and Proliferation

Anti-mouse CD28 recombinant proteins are primarily used to stimulate T cell activation in both in vitro and in vivo settings. When combined with anti-CD3 antibodies, these proteins effectively induce T cell proliferation and enhance immune responses. This dual stimulation approach (anti-CD3 plus anti-CD28) has become a standard protocol for reliable and reproducible T cell activation in research applications.

Immune Response Enhancement

CD28 signaling amplifies the overall immune response by enhancing T cell survival, proliferation, and cytokine production. The CD28/B7 interaction pathway prevents apoptosis of activated T cells through upregulation of anti-apoptotic factors, making these proteins valuable for studies examining T cell persistence and function.

Regulatory T Cell Expansion

Certain anti-CD28 antibodies function as superagonists and are commonly used to induce expansion of regulatory T cells (Tregs) in various mouse disease models. This application is particularly valuable for research into immune tolerance and inflammatory disease mechanisms.

Long-term Immune Memory Studies

CD28 signaling is essential for maintenance of long-term antigen-specific antibody production and persistence of plasma cells in bone marrow. This makes anti-CD28 recombinant proteins useful for investigating sustained immune responses and memory formation.

Technical Advantages

These recombinant proteins are typically formulated in phosphate-buffered saline with minimal endotoxin contamination (≤1 EU/mg), ensuring they won't confound experimental results with inflammatory artifacts. They can be used in multiple experimental formats including flow cytometry, functional assays, and in vivo studies, providing flexibility for diverse research designs.

You can use a recombinant protein as a standard for quantification or calibration in ELISA assays, provided it is highly purified, accurately quantified, and matches the analyte you intend to measure. For quantification of mouse CD28, the recombinant protein must be the same molecule (mouse CD28) and should be prepared in a suitable buffer compatible with your assay.

Key requirements and considerations:

• Purity and Quantification: The recombinant protein should be highly purified (typically ≥95%) and its concentration must be accurately determined, ideally by methods such as HPLC or spectrophotometry.
• Identity and Sequence: The recombinant protein must match the native mouse CD28 in sequence and structure, especially in the epitope recognized by your ELISA antibodies. Differences in folding or sequence can affect antibody recognition and quantification accuracy.
• Standard Curve Preparation: Prepare a serial dilution of the recombinant protein to generate a standard curve, covering the expected concentration range in your samples (commonly 0–1000 pg/mL, but may vary).
• Validation: Confirm that your ELISA antibodies recognize the recombinant protein with the same affinity as the native protein in your samples. This is critical for accurate quantification.
• Buffer Compatibility: Ensure the recombinant protein is reconstituted or diluted in a buffer compatible with your ELISA system, avoiding components that may interfere with antibody binding or detection.

Limitations and caveats:

• If the recombinant protein is an antibody (e.g., anti-mouse CD28), it cannot serve as a standard for quantifying mouse CD28 protein. Only the antigen (mouse CD28 protein) should be used as a standard for quantification of CD28.
• If your recombinant protein is an anti-mouse CD28 antibody (not the CD28 antigen), it is not suitable as a standard for quantifying CD28 in ELISA. Standards must be the same molecule as the analyte being measured.

Summary Table:

If your "Anti-Mouse CD28 Recombinant Protein" is actually an antibody, it cannot be used as a standard for quantifying CD28 protein in ELISA. If it is the recombinant mouse CD28 antigen, it can be used as a standard, provided the above criteria are met. Always verify the identity and intended use of your recombinant protein before proceeding.

Anti-Mouse CD28 Recombinant Protein has been validated for several key applications in published research, primarily involving T cell stimulation/activation both in vivo and in vitro, as well as flow cytometry, blocking/neutralization, and functional assays.

Validated Applications:

• In vivo T cell stimulation/activation: Used to induce T cell activation and proliferation, particularly for expanding regulatory T cells (Tregs) in mouse models of disease.
• In vitro T cell stimulation/activation: Applied to cultured mouse T cells to study costimulatory signaling, cytokine production (e.g., IL-2), and proliferation.
• Flow Cytometry (FC): Used to detect and quantify CD28 expression on mouse T cells and other immune subsets.
• Blocking/Neutralization: Employed to block CD28-mediated costimulatory signals, allowing investigation of CD28’s role in immune responses.
• Functional Assays: Utilized to assess T cell function, including proliferation, differentiation, and cytokine secretion in response to CD28 engagement.
• Agonist Activity: Certain clones act as CD28 agonists, directly stimulating T cells in the presence or absence of anti-CD3 antibodies.
• CyTOF-ready: Some recombinant anti-CD28 antibodies are validated for use in mass cytometry (CyTOF) after appropriate conjugation.

Supporting Details:

• Mechanistic studies: Anti-mouse CD28 recombinant proteins are frequently used to dissect the costimulatory role of CD28 in T cell biology, including its interaction with CD80/CD86 and downstream signaling pathways.
• Disease models: These reagents are applied in mouse models of autoimmunity, allergy, transplantation, and cancer to modulate T cell responses and study immune regulation.
• Combination protocols: Often used in combination with anti-CD3 antibodies to achieve robust T cell activation in vitro and in vivo.

Summary Table of Validated Applications

These applications are widely reported in peer-reviewed literature and product validation data, supporting the use of anti-mouse CD28 recombinant proteins in diverse immunological research settings.

To reconstitute and prepare Anti-Mouse CD28 Recombinant Protein for cell culture experiments, use sterile technique and follow these steps:

1. Reconstitution

• Lyophilized recombinant mouse CD28 protein is typically reconstituted in sterile PBS (phosphate-buffered saline).
• The recommended concentration for reconstitution is 200–400 μg/mL in sterile PBS. Some formulations may require the addition of at least 0.1% serum albumin (human or bovine) to stabilize the protein, especially if the product contains no carrier protein.
• Allow the vial and buffer to equilibrate to room temperature before opening. Centrifuge the vial briefly to collect all powder at the bottom.
• Add the appropriate volume of sterile PBS (with or without albumin, as specified) directly to the vial. Gently mix by inversion or slow pipetting. Avoid vigorous vortexing to prevent protein denaturation.
• Let the solution stand at room temperature for 15–30 minutes with gentle agitation to ensure complete dissolution. If visible particulates remain, continue gentle mixing for up to 2 hours.

2. Storage After Reconstitution

• After reconstitution, the protein can be stored at 2–8 °C for up to 1 month under sterile conditions.
• For longer-term storage, aliquot and freeze at –20 °C to –70 °C in a manual defrost freezer. Avoid repeated freeze-thaw cycles to maintain protein integrity.
• If the protein is used for functional assays, avoid adding sodium azide or other preservatives that may affect cell viability.

3. Preparation for Cell Culture Experiments

• Before use, dilute the reconstituted protein to the desired working concentration using sterile cell culture medium or PBS.
• Typical working concentrations for T cell activation assays are in the range of 0.3–5 μg/mL, but the optimal concentration should be determined empirically for your specific assay.
• For plate-bound activation, coat tissue culture plates with the protein (e.g., overnight at 4 °C), then wash to remove unbound protein before adding cells.
• For soluble stimulation, add the protein directly to the cell culture medium at the desired final concentration.

Best Practices

• Always use aseptic technique to prevent contamination.
• Use low-protein binding tubes and pipette tips to minimize protein loss.
• If the protein is sensitive or contains no carrier, consider adding 0.1% BSA to all buffers to prevent adsorption to plastic surfaces.

Summary Table

If your product datasheet specifies a different buffer or concentration, always follow the manufacturer’s instructions for that specific lot.