Recombinant Mouse Cathepsin B

Using Recombinant Mouse Cathepsin B in research applications is essential for studying the enzyme’s biological roles, disease mechanisms, and for developing or screening potential therapeutics, particularly in murine models.

Key reasons to use recombinant mouse Cathepsin B include:

• Functional Studies in Mouse Models: Cathepsin B is a lysosomal cysteine protease involved in protein turnover, apoptosis, autophagy, immune response, and cell migration. Using the recombinant mouse protein allows you to directly investigate these processes in a species-relevant context, which is critical for translational research and preclinical studies.

• Disease Mechanism Elucidation: Cathepsin B is implicated in a variety of diseases, including cancer, acute pancreatitis, cardiovascular disease, and viral infections (e.g., SARS-CoV-2). Recombinant protein enables controlled in vitro assays to dissect its role in these pathologies, such as its involvement in apoptosis, pyroptosis, and tumor invasion/metastasis.

• Enzyme Activity Assays and Inhibitor Screening: Recombinant Cathepsin B is used to measure enzymatic activity, substrate specificity, and to screen for inhibitors or activators, which is vital for drug discovery and mechanistic enzymology. Its activity can be quantified using fluorogenic peptide substrates, facilitating high-throughput screening.

• Pathway and Protein Interaction Studies: Cathepsin B participates in the activation of other proteases (e.g., trypsinogen, caspases), antigen processing for MHC class II presentation, and regulation of autophagy-lysosome pathways. Recombinant protein allows for precise biochemical and cell-based assays to map these interactions.

• High Purity and Consistency: Recombinant preparations offer high purity (≥95%), low endotoxin levels, and batch-to-batch consistency, which are critical for reproducible results in sensitive assays.

• Versatility in Experimental Design: Recombinant Cathepsin B can be used in a variety of applications, including:

  • Enzyme kinetics and substrate profiling
  • Cell culture experiments (e.g., to study protease-mediated signaling)
  • ELISA standards and bioassays
  • Protein-protein interaction studies

• Species-Specific Insights: Using the mouse version is crucial when your research involves murine cells, tissues, or in vivo models, ensuring biological relevance and avoiding cross-species differences that may confound results.

In summary, recombinant mouse Cathepsin B is a powerful tool for dissecting its physiological and pathological roles in mouse systems, supporting both basic research and translational applications such as drug screening and disease modeling.

Yes, recombinant Mouse Cathepsin B protein can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated and matched to the assay system.

Most commercial Mouse Cathepsin B ELISA kits use recombinant Mouse Cathepsin B as their standard for generating calibration curves. The recombinant protein is serially diluted to create a standard curve, which is then used to interpolate the concentration of Cathepsin B in unknown samples. This approach is standard practice in quantitative ELISA protocols.

Key considerations for using recombinant Mouse Cathepsin B as a standard:

• The recombinant protein should be of high purity and accurately quantified.
• It should match the epitope recognized by the antibodies in your ELISA kit (i.e., same sequence and post-translational modifications, if relevant).
• The standard should be reconstituted and stored according to the manufacturer’s instructions to maintain stability and activity.
• Serial dilutions must be prepared in the same buffer as your samples to minimize matrix effects.
• Always run a standard curve with each assay to ensure accuracy and reproducibility.

Typical protocol steps:

• Prepare serial dilutions of recombinant Mouse Cathepsin B in assay diluent to cover the dynamic range of your ELISA (e.g., 156–10,000 pg/mL).
• Include a zero standard (diluent only) for baseline correction.
• Plot the standard curve (OD vs. concentration) and interpolate sample concentrations from this curve.

Assay specificity:
Validated ELISA kits detect both endogenous and recombinant Mouse Cathepsin B, with no significant cross-reactivity to other proteins.

Summary:
Using recombinant Mouse Cathepsin B as a standard is scientifically appropriate and widely accepted for ELISA quantification, as long as the protein is compatible with your assay system and handled according to best practices.

Recombinant Mouse Cathepsin B has been validated across multiple research applications in published literature, demonstrating its utility as a functional protease reagent in diverse experimental contexts.

Enzyme Activity and Bioassay Applications

The protein has been extensively used in bioassay applications to measure enzymatic activity. Functional validation typically employs fluorogenic peptide substrates, such as Z-LR-AMC, to quantify proteolytic activity. This capability has enabled researchers to investigate cathepsin B's role in various cellular processes and disease mechanisms.

Immunological and Cellular Research

Recombinant mouse cathepsin B has been applied in studies examining immune cell function and cellular signaling. Published research has utilized this protein in whole cell bioassays to investigate cathepsin B's involvement in macrophage necroptosis, where the protease cleaves Rip1 kinase to modulate cell death pathways. Additionally, studies have employed the protein to examine cathepsin B's role in myeloid-derived suppressor cell function and inflammasome activation in tumor microenvironments.

Neurodegenerative Disease Models

Recent research has validated recombinant cathepsin B in models of synucleinopathy, demonstrating that the protein effectively reduces α-synuclein levels and improves lysosomal function in neuronal cells. Treatment with recombinant cathepsin B recovered β-glucocerebrosidase activity and reduced synuclein-dependent synaptic defects, suggesting therapeutic potential for Parkinson's disease and related conditions. The protein has also been investigated in traumatic brain injury models, where cathepsin B activity contributes to neurological deficits.

Immunohistochemistry and Detection

The protein serves as a positive control and reference standard for immunohistochemical detection of endogenous cathepsin B in tissue sections. This application validates antibody specificity and enables localization studies of the protease in various tissue types.

Protein Degradation Pathway Studies

Recombinant mouse cathepsin B has been utilized to investigate the lysosomal protein degradation and autophagy-lysosome pathways, supporting research into cellular recycling mechanisms and energy homeostasis.

To reconstitute and prepare Recombinant Mouse Cathepsin B protein for cell culture experiments, first centrifuge the vial briefly, then reconstitute the lyophilized protein in sterile distilled water or PBS (pH 7.2–7.4) to a concentration of 0.1–0.5 mg/mL, avoiding vigorous mixing or vortexing. For functional assays or activation, dilute the protein in an activation buffer (e.g., 25 mM MES, 5 mM DTT, pH 5.0), incubate at room temperature for 15 minutes, then further dilute as needed for your experiment.

Step-by-step protocol:

• Centrifuge the vial briefly (10,000 × g for 30 seconds) before opening to ensure all protein is at the bottom.
• Reconstitute the lyophilized protein in sterile distilled water or PBS (pH 7.2–7.4) to a final concentration of 0.1–0.5 mg/mL. Do not use vortexing or vigorous pipetting; gently mix by inverting or slow pipetting.
• If required for activation, dilute the protein to 10 µg/mL in activation buffer (25 mM MES, 5 mM DTT, pH 5.0).
• Incubate at room temperature for 15 minutes to activate the enzyme.
• Dilute further in assay or cell culture buffer as needed for your experiment. For cell culture, ensure the buffer is compatible with your cells and does not contain cytotoxic components (e.g., avoid high DTT concentrations unless required for activity and validated for cell compatibility).
• Aliquot and store any unused reconstituted protein at 4–8°C for up to 2–7 days, or at –20°C for longer-term storage. Avoid repeated freeze/thaw cycles.

Additional notes:

• The optimal pH for Cathepsin B activity is between 4 and 6.
• For cell culture experiments, confirm the absence of endotoxin and other contaminants if using sensitive cell types.
• Always consult the specific product datasheet for recommended buffers and concentrations, as requirements may vary by protein preparation.

Summary of key points:

• Reconstitute in sterile water or PBS, pH 7.2–7.4.
• Avoid vigorous mixing.
• Activate in MES/DTT buffer if required.
• Store aliquots appropriately.
• Use compatible buffers for cell culture.