Recombinant Mouse Granzyme B

Recombinant Mouse Granzyme B is widely used in research because it is a key effector protease in immune-mediated cell death, making it essential for studying cytotoxic lymphocyte function, immune regulation, and disease mechanisms involving apoptosis and inflammation.

Key reasons to use recombinant mouse Granzyme B in research applications:

• Mechanistic Studies of Apoptosis: Granzyme B is a serine protease released by cytotoxic T lymphocytes and NK cells to induce apoptosis in target cells by activating caspases and cleaving other substrates, such as BID, leading to DNA fragmentation and cell death. Using recombinant protein allows precise control over experimental conditions to dissect these pathways.

• Immune Response and Disease Modeling: Granzyme B is central to immune surveillance, eliminating virally infected, transformed, or allogeneic cells. Its role extends to autoimmune diseases, inflammatory disorders, cancer immunotherapy, and transplantation biology, making it a versatile tool for modeling these processes in vitro and in vivo.

• Biomarker and Functional Assays: Granzyme B serves as a biomarker for T cell-mediated cytotoxicity and immune activation, useful in monitoring immune responses in disease models or therapeutic interventions.

• Species-Specific Functional Analysis: Recombinant mouse Granzyme B is necessary for studies in murine systems due to differences in substrate specificity and activity compared to human Granzyme B. This is critical for accurate modeling of mouse immune responses and for cross-species comparison studies.

• Therapeutic Research and Drug Screening: Granzyme B is a target for developing novel immunotherapies, including engineered cytolytic fusion proteins and small molecule inhibitors. Recombinant protein is essential for high-throughput screening and mechanistic validation.

• Biochemical and Structural Studies: Recombinant protein enables detailed enzymatic assays, substrate profiling, and structural analyses, supporting drug discovery and fundamental research.

Technical advantages of recombinant protein:

• High purity and defined activity, minimizing variability.
• Absence of contaminating proteases or immune factors.
• Amenable to modification (e.g., tags, mutations) for mechanistic or structural studies.

In summary, recombinant mouse Granzyme B is indispensable for immunology, oncology, and cell death research, providing a reliable, species-specific tool for dissecting cytotoxic mechanisms, modeling disease, and developing targeted therapies.

Yes, recombinant mouse Granzyme B can be used as a standard for quantification or calibration in ELISA assays, provided it is compatible with your assay system and matches the native protein's immunoreactivity. Most commercial mouse Granzyme B ELISA kits use recombinant mouse Granzyme B as the standard to generate the calibration curve for quantification.

Key considerations and supporting details:

• ELISA Standard Curve: The standard curve in quantitative ELISA is typically generated using a recombinant form of the target protein with a known concentration, allowing for accurate quantification of the analyte in unknown samples.
• Kit Compatibility: ELISA kits for mouse Granzyme B are validated to recognize both native and recombinant forms of the protein, ensuring that recombinant standards are suitable for calibration.
• Standard Preparation: When using recombinant mouse Granzyme B as a standard, it is essential to prepare it in the same buffer or diluent as recommended by your ELISA protocol to minimize matrix effects and ensure accurate quantification.
• Immunoreactivity: The recombinant standard should be full-length and properly folded to ensure it is recognized by the capture and detection antibodies in the assay, as some kits specify recognition of both native and recombinant forms.
• Documentation: Many ELISA kit manuals explicitly state that their standard is recombinant mouse Granzyme B, and some vendors offer recombinant protein specifically for use as an ELISA standard.

Best Practices:

• Confirm that your recombinant mouse Granzyme B is of high purity and its concentration is accurately determined.
• Validate the linearity and recovery of your standard curve using the recombinant protein in your specific assay matrix.
• If using a recombinant standard not supplied with your ELISA kit, ensure it is compatible with the antibodies used in your assay (e.g., same isoform, post-translational modifications if relevant).

Summary Table: Use of Recombinant Mouse Granzyme B as ELISA Standard

In conclusion, recombinant mouse Granzyme B is widely accepted and routinely used as a standard for quantification in ELISA assays, provided it is compatible with your assay system and prepared according to the kit instructions.

Recombinant Mouse Granzyme B has been validated for several key applications in published research, primarily in studies of apoptosis, immune cell cytotoxicity, and therapeutic intervention models.

Validated Applications:

• Induction of Apoptosis in Target Cells: Recombinant mouse Granzyme B is widely used to simulate the cytotoxic effects of NK cells and cytotoxic T lymphocytes (CTLs) on target cells, particularly by inducing apoptosis via caspase activation and Bid cleavage pathways. This is central to studies investigating immune-mediated cell death.

• In Vitro Cytotoxicity Assays: It is applied to cell cultures to directly assess its ability to induce apoptosis, often measured by caspase activation, DNA fragmentation, or cell viability assays. For example, in liver transplantation models, recombinant Granzyme B was added to hepatocyte cultures to mimic NK cell-mediated cytotoxicity and study apoptosis during ischemia-reperfusion injury.

• Biochemical Activity Assays: The protein is validated for enzymatic assays using synthetic substrates (e.g., Boc-AAD-SBzl in the presence of DTNB) to measure its protease activity, which is crucial for characterizing its function and for inhibitor screening.

• Therapeutic Target Validation: Recombinant Granzyme B is used in preclinical models to evaluate potential inhibitors and therapeutic strategies, such as testing small molecules that block its activity in disease models (e.g., liver injury, cancer immunotherapy).

• Immunology and Oncology Research: It serves as a biomarker and functional reagent in studies of immune cell-mediated cytotoxicity, tumor cell killing, and immune response modulation.

Supporting Details:

• Mechanistic Studies: Recombinant Granzyme B is used to dissect molecular pathways of apoptosis, including caspase-dependent and mitochondrial pathways, by direct addition to cell lysates or intact cells.
• Inhibitor Screening: Its validated enzymatic activity enables high-throughput screening of candidate inhibitors for therapeutic development.
• Functional Reconstitution: Adoptive transfer experiments and cell culture models use recombinant Granzyme B to restore or simulate cytotoxic function in genetically modified or depleted immune cells.

Additional Notes:

• Recombinant mouse Granzyme B is typically produced as a mature, active protein and validated for purity and activity using biochemical and cell-based assays.
• It is not generally used for antibody-based detection (e.g., flow cytometry or immunohistochemistry), but rather as a functional protein for mechanistic and therapeutic studies.

In summary, recombinant mouse Granzyme B is validated for use in apoptosis induction, cytotoxicity assays, biochemical activity measurements, therapeutic target validation, and mechanistic immunology research. These applications are well-supported in published literature and are central to studies of immune cell function and cell death pathways.

To reconstitute and prepare Recombinant Mouse Granzyme B protein for cell culture experiments, follow these best-practice steps based on protein handling protocols and specific recommendations for granzyme B:

1. Equilibrate and Centrifuge the Vial

• Allow the lyophilized protein vial and your chosen reconstitution buffer to reach room temperature (18–25°C) before opening to minimize condensation and ensure accurate reconstitution.
• Briefly centrifuge the vial to collect all powder at the bottom before opening.

2. Choose an Appropriate Reconstitution Buffer

• For most cell culture applications, use sterile water or sterile PBS (phosphate-buffered saline), pH 7.2–7.4, unless your protocol specifies otherwise.
• If the protein is to be used in enzymatic assays or requires activation, refer to specific buffer requirements (e.g., activation buffer with cathepsin C for enzymatic activity).

3. Reconstitute the Protein

• Add sterile water or buffer to achieve a final concentration of 0.1–1.0 mg/mL (typical range for recombinant proteins).
• For example, to reconstitute 10 μg of protein to 1.0 mg/mL, add 10 μL of buffer; for 0.1 mg/mL, add 100 μL.
• Gently swirl or invert the vial to dissolve. Avoid vigorous vortexing or pipetting to prevent foaming and protein denaturation.
• Let the vial stand at room temperature for 15–30 minutes with gentle agitation. If undissolved material remains, continue gentle mixing for up to 2 hours.

4. Aliquot and Storage

• Once fully dissolved, aliquot the protein into single-use volumes to avoid repeated freeze-thaw cycles, which can degrade protein activity.
• Store aliquots at –20°C or –80°C. For short-term use (days), 4°C is acceptable, but avoid prolonged storage at this temperature.

5. Preparation for Cell Culture

• If using in cell culture, dilute the reconstituted stock to the desired working concentration in cell culture medium immediately before use.
• If the protein requires activation (e.g., with cathepsin C), follow the activation protocol before adding to cells.
• Filter-sterilize the final working solution if sterility is required and the buffer is compatible with filtration.

6. Additional Notes

• Avoid repeated freeze-thaw cycles by using aliquots.
• If the protein is sensitive or prone to aggregation, consider adding a carrier protein (e.g., 0.1% BSA) to the buffer for stabilization, unless this interferes with your assay.
• Always consult the specific datasheet for your recombinant protein for any unique requirements.

Summary Table: Key Steps for Reconstitution

These steps ensure optimal solubility, stability, and activity of recombinant mouse granzyme B for cell culture experiments. Always verify with the specific product datasheet for any unique instructions.