Recombinant Human Pro-Caspase-3

Recombinant Human Pro-Caspase-3 is used in research applications to study the molecular mechanisms of apoptosis, screen for apoptosis-modulating compounds, and investigate caspase activation pathways in vitro. It provides a controlled, defined system for activating and analyzing caspase-3 function, which is central to programmed cell death and related cellular processes.

Key reasons to use Recombinant Human Pro-Caspase-3 in research:

• Modeling Apoptosis Activation: Pro-caspase-3 is the inactive precursor of caspase-3, the major executioner caspase in apoptosis. By using the recombinant pro-form, researchers can precisely control its activation (e.g., by adding initiator caspases like caspase-8 or chemical activators), allowing detailed study of the activation process and downstream effects.
• Enzyme Kinetics and Substrate Specificity: Recombinant pro-caspase-3 enables kinetic assays to measure activation rates, substrate cleavage, and inhibitor screening under defined conditions, which is essential for drug discovery and mechanistic studies.
• Pathway Dissection: Using the pro-form allows researchers to dissect upstream and downstream signaling events in apoptosis, distinguishing between effects on caspase activation versus effects on active caspase-3 function.
• Standardization and Reproducibility: Recombinant proteins provide batch-to-batch consistency, purity, and defined concentration, which are critical for reproducible biochemical assays and quantitative studies.
• Disease Modeling: Dysregulation of caspase-3 is implicated in cancer, neurodegeneration, and inflammatory diseases. Recombinant pro-caspase-3 is used to model these processes in vitro and to test therapeutic interventions targeting the apoptotic pathway.

Typical applications include:

• In vitro activation assays to study caspase-3 processing and function.
• Screening for caspase inhibitors or activators for therapeutic development.
• Elucidating the role of caspase-3 in cell death, inflammation, and disease models.
• Serving as a standard or control in ELISA, Western blot, or mass cytometry assays.

Summary:
Using recombinant human pro-caspase-3 allows for precise, reproducible, and mechanistically informative studies of apoptosis and related cellular processes, supporting both basic research and drug discovery efforts.

Recombinant Human Pro-Caspase-3 can potentially be used as a standard for quantification or calibration in ELISA assays, but only if the ELISA is designed to detect the pro-form and not the active (cleaved) form of caspase-3. The suitability depends on the specificity of the antibodies used in your ELISA and the intended analyte.

Key considerations:

• Form of Caspase-3 Detected: Most commercial ELISA kits for caspase-3 are designed to detect either the total, active (cleaved), or specifically the pro-form of caspase-3. If your ELISA is specific for the active (cleaved) form, using pro-caspase-3 as a standard will not yield accurate quantification, as the antibodies may not recognize the pro-form equivalently. If your ELISA detects total caspase-3 (both pro and active forms), the recombinant pro-caspase-3 may be suitable, but this must be confirmed by the kit documentation or by validation experiments.

• Standard Preparation: Some ELISA kits use recombinant active caspase-3 as the standard, while others may use full-length or pro-caspase-3. The standard must match the form of caspase-3 that your assay is designed to detect for accurate calibration.

• Carrier Protein: Recombinant pro-caspase-3 is available in carrier-free and BSA-containing formulations. For ELISA standards, the BSA-containing version is generally recommended to enhance stability and mimic sample matrix effects, unless BSA interferes with your assay.

• Validation: If your ELISA kit does not specify the use of pro-caspase-3 as a standard, you must validate its performance by generating a standard curve and confirming parallelism with endogenous caspase-3 in your sample matrix.

Summary Table: Pro-Caspase-3 as ELISA Standard

Best Practices:

• Check your ELISA kit documentation for the form of caspase-3 used as the standard and the specificity of the capture/detection antibodies.
• Validate the standard curve using recombinant pro-caspase-3 to ensure it matches the response of endogenous caspase-3 in your samples.
• Use the BSA-containing formulation for standards unless BSA interferes with your assay.

If your ELISA is designed for active caspase-3, you should use a recombinant active caspase-3 standard instead. If it is for pro-caspase-3 or total caspase-3 and the antibodies recognize the pro-form, recombinant human pro-caspase-3 is appropriate as a standard.

Recombinant Human Pro-Caspase-3 has been validated primarily for bioassays measuring enzyme activity, including studies of apoptotic proteolysis, substrate cleavage, and activation mechanisms in published research.

Key validated applications include:

• Enzyme Activity Assays: Used to quantify caspase-3 activation and measure its proteolytic activity on specific substrates, often employing fluorescent plate readers and kinetic measurements. This includes protocols where pro-caspase-3 is activated (e.g., by caspase-8) and its activity is measured using synthetic substrates.

• Substrate Cleavage Studies: Validated for investigating cleavage of physiological substrates such as PARP, proIL-16, PKC isoforms, procaspases 6, 7, and 9, and β-catenin, which are relevant to apoptosis research.

• Cell Lysate Bioassays: Applied to cell lysates to study caspase-3 activation and downstream effects in cellular models of apoptosis and inflammation.

• Mechanistic Studies of Apoptosis: Used to dissect the molecular pathways of apoptosis, including intrinsic and extrinsic signaling, and to study the role of caspase-3 in cell death and inflammation.

Additional research applications (as supported by published literature):

• Cancer Biology: Utilized in studies examining caspase-3’s role in oncogene-induced malignant transformation and tumor progression, including modulation of signaling pathways such as Src-STAT3.

• Drug Screening and Inhibitor Validation: Employed in the validation of caspase-3-selective inhibitors and in dose- and time-dependent experiments to define caspase-3’s cellular roles.

• Neurodegeneration Models: Engineered variants (e.g., light-activated caspase-3) have been used in model organisms to study neurodegeneration and cell ablation, leveraging the conserved substrate specificity of human caspase-3.

Summary Table: Validated Applications

Note: Most published protocols use recombinant pro-caspase-3 in in vitro biochemical assays, cell-based assays, and mechanistic studies of apoptosis. For specific experimental details, refer to the cited research articles and product datasheets.

To reconstitute and prepare Recombinant Human Pro-Caspase-3 protein for cell culture experiments, follow these general guidelines based on manufacturer protocols and best practices for recombinant proteins:

1. Reconstitution

• Buffer: Reconstitute the lyophilized protein in sterile, cold buffer. Commonly used buffers include:
  • 20 mM Tris, 300 mM NaCl, 5 mM DTT, 5% sucrose, 0.05% CHAPS, pH 8.0 (as recommended by R&D Systems for pro-caspase-3).
  • Alternatively, 10 mM PBS (pH 7.4) or phosphate-buffered saline (PBS) can be used, depending on the manufacturer’s instructions.
• Concentration: Reconstitute to a concentration of 0.1–1.0 mg/mL (or as specified by the product datasheet).
• Procedure:
  • Briefly centrifuge the vial before opening to bring the contents to the bottom.
  • Add the appropriate volume of buffer to achieve the desired concentration.
  • Gently mix by swirling or pipetting up and down. Do not vortex, as this may denature the protein.
  • Allow the protein to dissolve at room temperature for 10–15 minutes, mixing gently if needed.

2. Storage

• Short-term: Store reconstituted protein at 4°C for up to a few days if used immediately.
• Long-term: Aliquot and store at –80°C to avoid repeated freeze-thaw cycles, which can reduce activity.
• Lyophilized protein: Store desiccated at –20°C to –70°C.

3. Preparation for Cell Culture

• Buffer Exchange (if needed): If the reconstitution buffer contains components not compatible with cell culture (e.g., CHAPS, high salt, or DTT), dialyze or desalt the protein into a cell-compatible buffer such as PBS or serum-free culture medium.
• Sterility: Ensure all steps are performed under sterile conditions to avoid contamination.
• Concentration Adjustment: Dilute the protein to the desired working concentration in culture medium or buffer just before use.

4. Handling Tips

• Avoid repeated freeze-thaw cycles.
• Mix gently to prevent protein aggregation.
• Confirm protein concentration using a suitable assay (e.g., Bradford or BCA).

5. Application in Cell Culture

• Add the reconstituted and diluted pro-caspase-3 to cells at the desired concentration, typically in the range of 1–10 µg/mL, depending on the experimental design.
• Monitor cell viability and caspase activity as appropriate.

Always refer to the specific product datasheet for exact reconstitution and handling instructions, as protocols may vary slightly between manufacturers.