Recombinant Human Cathepsin X/Z/P

Recombinant Human Cathepsin X/Z/P is a valuable tool for research applications due to its unique enzymatic properties and broad biological relevance. Here are several reasons why you should consider using it in your research:

1. Unique Enzymatic Activity

Cathepsin X/Z/P is a cysteine protease that functions as a carboxypeptidase, meaning it cleaves amino acids from the C-terminus of peptides and proteins. This distinguishes it from other cathepsins, which typically act as endopeptidases. Its unique specificity allows for targeted studies of carboxypeptidase activity and substrate processing in biological systems.

2. High Purity and Carrier-Free Format

Recombinant Human Cathepsin X/Z/P is available with >95% purity and in a carrier-free format (without BSA). This is particularly important for applications where the presence of carrier proteins could interfere, such as enzyme kinetics, structural studies, or when precise quantification of the enzyme is required.

3. Broad Biological Relevance

Cathepsin X/Z/P is ubiquitously expressed in human tissues and is conserved across species. It plays roles in various physiological processes, including:

• Protein turnover and degradation
• Immune response modulation
• Cell signaling
• Tissue remodeling

Its involvement in these processes makes it a relevant target for studying diseases such as cancer, neurodegenerative disorders, and inflammatory conditions.

4. Dimeric Structure

Recent studies have shown that Cathepsin X/Z/P exists as a biologically active homodimer. This dimeric structure may influence its enzymatic activity and substrate specificity, providing new insights into its function and potential regulatory mechanisms.

5. Versatile Applications

Recombinant Human Cathepsin X/Z/P can be used in a variety of research applications, including:

• Enzyme activity assays
• Substrate specificity studies
• Inhibitor screening
• Structural and functional characterization
• Cell-based assays

6. Therapeutic and Diagnostic Potential

Cathepsin X/Z/P has been implicated in several pathological conditions, making it a potential biomarker and therapeutic target. Research using recombinant Cathepsin X/Z/P can contribute to the development of selective inhibitors and diagnostic tools for diseases where cathepsin activity is dysregulated.

7. Standardized and Reliable Source

Recombinant proteins provide a consistent and reliable source of enzyme, ensuring reproducibility in experiments. This is crucial for comparative studies and for validating findings across different laboratories.

In summary, Recombinant Human Cathepsin X/Z/P is a versatile and well-characterized enzyme that can enhance your research by providing insights into carboxypeptidase activity, substrate specificity, and the biological roles of cathepsins in health and disease.

Yes, recombinant Human Cathepsin X/Z/P can be used as a standard for quantification or calibration in ELISA assays, provided it is sufficiently pure and its concentration is accurately determined.

For ELISA standard curve generation, the following technical considerations are essential:

• Purity and Characterization: The recombinant protein should be highly purified, ideally >80% as confirmed by SDS-PAGE or similar methods. Impurities can affect assay accuracy.
• Concentration Determination: The protein concentration must be precisely measured, typically by absorbance at 280 nm, BCA assay, or HPLC. Lot-specific datasheets should be consulted for accurate concentration information.
• Carrier Protein: Recombinant Cathepsin X/Z/P is available in both carrier-free and BSA-containing formulations. For ELISA standards, BSA-containing formulations are generally recommended to enhance stability and prevent adsorption to plasticware. However, if BSA interferes with your assay, use the carrier-free version.
• Reconstitution and Storage: Follow manufacturer or supplier instructions for reconstitution and storage. Lyophilized standards should be reconstituted carefully, and aliquots should be stored at -20°C to -70°C to maintain activity and stability.
• Validation: The recombinant standard should be validated in your specific ELISA format (e.g., sandwich ELISA) to ensure it behaves similarly to the native protein in your samples. Some recombinant standards are specifically validated for ELISA applications, as indicated in product documentation.

Protocol Best Practices:

• Prepare a serial dilution of the recombinant Cathepsin X/Z/P to cover the expected concentration range in your samples (commonly 0–1000 pg/mL, but may vary).
• Use curve-fitting software (e.g., four-parameter logistic regression) to generate the standard curve.
• Include appropriate controls and blanks to account for background signal and non-specific binding.

Limitations and Considerations:

• The recombinant protein may differ from endogenous Cathepsin X/Z/P in post-translational modifications or folding, which can affect antibody recognition in some ELISA formats. Confirm compatibility with your capture and detection antibodies.
• If using a recombinant standard not specifically validated for your ELISA kit, results may vary due to differences in calibration and technique.

In summary, recombinant Human Cathepsin X/Z/P is suitable as an ELISA standard if it is pure, accurately quantified, and validated for your assay conditions. Always consult technical datasheets and perform preliminary validation in your assay system.

Recombinant Human Cathepsin X/Z/P has been validated in published research for a range of applications, primarily focused on its enzymatic activity, role in disease models, and use as a standard or control in various assay formats.

Validated Applications in Published Research:

• Enzyme Activity Assays:
  Used to characterize its carboxypeptidase activity, substrate specificity, and inhibitor profiling in biochemical assays. These studies often employ recombinant protein to measure kinetic parameters or to screen for selective inhibitors relevant to cancer and neurodegenerative disease research.

• Bioassays:
  Applied in functional bioassays to study its biological effects, such as its role in cell migration, neurite outgrowth, and proteolytic processing of substrates. For example, recombinant Cathepsin X has been used to demonstrate its involvement in tumor cell migration and neuritogenesis in cell-based models.

• ELISA Standard:
  Utilized as a standard in the development and validation of ELISA assays for quantifying Cathepsin X/Z/P in biological samples, including serum from cancer patients. This enables the assessment of its prognostic and predictive value in clinical studies.

• Western Blot (WB):
  Used as a positive control or for antibody validation in Western blotting to detect Cathepsin X/Z/P in cell lysates and tissue extracts. This application is essential for confirming protein expression and antibody specificity.

• Immunohistochemistry (IHC) and Immunocytochemistry (ICC):
  Employed as a control or for antigen retrieval optimization in IHC and ICC protocols, particularly in studies examining Cathepsin X/Z/P localization in normal and cancerous tissues. Both paraffin-embedded (IHC-P) and frozen (IHC-Fr) tissue sections have been analyzed.

• Flow Cytometry:
  Used in flow cytometric assays to study Cathepsin X/Z/P expression and function in whole cells, such as in macrophage invasion assays.

• Activity-Based Probe Validation:
  Recombinant Cathepsin X/Z/P has been used to validate activity-based probes designed to detect cysteine cathepsin activities in whole cells, confirming probe specificity and sensitivity.

Summary Table of Validated Applications

Additional Context:

• Recombinant Cathepsin X/Z/P is also used in mechanistic studies of its role in cancer progression, neurodegenerative diseases, and immune cell function.
• It serves as a tool for developing and validating selective inhibitors, which are of interest for therapeutic research.
• The protein is often produced in mammalian or insect cell systems to ensure proper folding and post-translational modifications relevant for functional studies.

These applications are well-supported in the literature and demonstrate the versatility of recombinant Cathepsin X/Z/P in both basic and translational research contexts.

To reconstitute and prepare Recombinant Human Cathepsin X/Z/P protein for cell culture experiments, follow these steps based on best practices for lyophilized recombinant proteins and specific protocols for cathepsins:

1. Reconstitution of Lyophilized Protein

• Briefly centrifuge the vial to collect all powder at the bottom before opening.
• Reconstitute the protein in sterile, deionized water or sterile PBS (pH 7.4) to your desired concentration (typically 0.1–1.0 mg/mL for stock solutions).
• Gently mix by pipetting or swirling. Avoid vigorous shaking or vortexing to prevent foaming and protein denaturation.
• Allow the vial to sit at room temperature for 15–30 minutes to ensure complete dissolution.

2. Activation of Cathepsin X/Z/P (if required for activity)

• Cathepsin X/Z/P is a cysteine protease and may require activation by reducing agents for full enzymatic activity.
• Prepare an Activation Buffer: 25 mM sodium acetate, pH 3.5.
• Prepare a DTT (dithiothreitol) stock solution (1 M in deionized water), then dilute to 10 mM in Activation Buffer.
• Dilute the reconstituted protein to 20 µg/mL in Activation Buffer.
• Mix equal volumes of 20 µg/mL protein and 10 mM DTT, and incubate at room temperature for 5 minutes to activate the enzyme.
• For cell culture applications where enzymatic activity is not required, this activation step may be omitted.

3. Preparation for Cell Culture

• After reconstitution (and activation if needed), dilute the protein into your cell culture medium to the desired working concentration.
• If the protein was reconstituted in a buffer incompatible with your cells (e.g., high DTT or low pH), perform a buffer exchange (e.g., by dialysis or desalting columns) into a physiological buffer such as PBS or directly into cell culture medium.
• Filter-sterilize the final solution using a 0.22 µm filter if sterility is required.

4. Storage

• Store unused reconstituted protein in aliquots at ≤–20 °C to avoid repeated freeze-thaw cycles.
• Short-term storage (2–7 days) can be at 4–8 °C.
• For long-term storage, consider adding 5–50% glycerol to prevent freeze-thaw damage.

5. Additional Notes

• Always consult the specific product datasheet for any unique requirements or stabilizers present in the formulation (e.g., trehalose, mannitol, Tween 80).
• For cell-based assays, ensure the final buffer composition is compatible with your cells and does not contain cytotoxic additives.

Summary Table: Key Steps

This protocol ensures optimal solubility, activity, and compatibility of recombinant Cathepsin X/Z/P for cell culture experiments.