Cleaved-CTSD (G65) Antibody

What is Cleaved-CTSD (G65) Antibody and what does it detect?

Cleaved-CTSD (G65) polyclonal antibody is an affinity-purified rabbit antibody that specifically detects endogenous levels of fragment of activated Cathepsin D light chain (LC) protein resulting from cleavage adjacent to G65. The antibody recognizes the human CATD protein at the amino acid range 46-95, making it a valuable tool for studying proteolytic processing of Cathepsin D in experimental settings . This antibody was produced against a synthesized peptide derived from the human CATD protein, and has been validated for use in Western Blot and ELISA applications with human and monkey samples .

What is the biological significance of Cathepsin D and its cleaved forms?

Cathepsin D (CTSD) is a member of the A1 family of peptidases that functions as an acid protease active in intracellular protein breakdown. The CTSD gene encodes a preproprotein that undergoes proteolytic processing to generate multiple protein products, including the cathepsin D light and heavy chains which heterodimerize to form the mature enzyme .

Functionally, Cathepsin D plays significant roles in:

• Intracellular protein breakdown and turnover

• APP processing following cleavage and activation by ADAM30, leading to APP degradation

• Pathogenesis of several diseases including breast cancer and possibly Alzheimer's disease

The protein undergoes complex post-translational modifications, including N- and O-glycosylation and proteolytic cleavage. The major heavy chain starts at Leu-169, with minor forms starting at Gly-170, Gly-171, and another form at Ala-168 .

How should Cleaved-CTSD (G65) Antibody be stored and handled to maintain its effectiveness?

For optimal maintenance of antibody effectiveness:

• Store the antibody at -20°C for up to 1 year from the date of receipt

• Avoid repeated freeze-thaw cycles, which can compromise antibody integrity

• The antibody is formulated as a liquid in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• Prior to use, allow the antibody to equilibrate to room temperature before opening the vial

The presence of glycerol in the formulation acts as a cryoprotectant, while BSA helps stabilize the antibody structure. Sodium azide serves as a preservative to prevent microbial contamination.

What are the recommended dilutions for different experimental applications?

Based on validation studies, the following dilution ranges are recommended:

These dilution guidelines have been established to provide optimal signal-to-noise ratios in the specified applications. For Western blot applications, optimization might be required depending on the sample type, protein loading, and detection system .

How can I confirm the specificity of Cleaved-CTSD (G65) Antibody in my experiments?

To confirm antibody specificity:

• Include appropriate positive controls (cell lines known to express Cathepsin D, such as COS7 cells treated with etoposide 25μM for 1h)

• Perform blocking experiments using the synthesized peptide derived from the human CATD at amino acid range 46-95

• Include negative controls such as non-relevant antibodies of the same isotype

• If possible, perform immunoprecipitation followed by mass spectrometry to confirm target identity

Western blot analysis has demonstrated that this antibody specifically recognizes the cleaved form of Cathepsin D, as evidenced by blocking experiments where the signal is abolished when the antibody is pre-incubated with the synthesized peptide .

How does the detection of cleaved CTSD (G65) differ from detection of total CTSD, and what are the implications for studying proteolytic processing?

Detecting cleaved CTSD (G65) versus total CTSD provides distinct insights into Cathepsin D processing:

The Cleaved-CTSD (G65) antibody specifically detects fragments of activated Cathepsin D LC protein resulting from cleavage adjacent to G65, focusing on the amino acid region 46-95 . This specificity allows researchers to investigate:

• The extent of proteolytic processing of Cathepsin D in various cellular conditions

• The accumulation of specific cleaved fragments under pathological conditions

• The temporal dynamics of CTSD processing during cellular responses

This distinction is critical when studying conditions where proteolytic processing may be altered, such as in Alzheimer's disease where CTSD processing following ADAM30-mediated cleavage influences APP degradation .

What is the relationship between Cathepsin D processing and its role in neurodegenerative diseases?

Cathepsin D processing has significant implications for neurodegenerative diseases:

CTSD is involved in the pathogenesis of several diseases, including potentially Alzheimer's disease . The protein plays a role in APP processing following cleavage and activation by ADAM30, which leads to APP degradation . Dysregulation of this process may contribute to the accumulation of pathogenic APP fragments.

Additionally, mutations in CTSD play a causal role in neuronal ceroid lipofuscinosis-10, a neurodegenerative lysosomal storage disorder . This highlights the importance of proper CTSD processing and function in maintaining neuronal health.

Research tracking cleaved forms of CTSD can provide insights into:

• Alterations in lysosomal function in neurodegenerative conditions

• Changes in proteolytic processing pathways

• Potential biomarkers for disease progression or therapeutic responses

How can I design experiments to investigate the relationship between CTSD cleavage and cellular localization?

To investigate CTSD cleavage in relation to cellular localization:

Experimental Design Strategy:

• Subcellular Fractionation Combined with Western Blotting:

  • Isolate distinct cellular compartments (lysosome, melanosome, secreted fraction, extracellular space)

  • Perform Western blotting with Cleaved-CTSD (G65) antibody on each fraction

  • Compare the distribution of cleaved forms across compartments

• Confocal Microscopy Co-localization:

  • Use Cleaved-CTSD (G65) antibody alongside organelle markers

  • Perform immunofluorescence staining with antibodies against:

    • LAMP1 (lysosomal marker)

    • Melanosome markers (if studying melanocytes)

    • Markers for extracellular vesicles

  • Quantify co-localization coefficients to determine predominant locations

• Live-Cell Imaging with Fluorescently Tagged CTSD:

  • Generate constructs with mutations at cleavage sites

  • Monitor trafficking and localization changes in real-time

This approach can reveal how different cellular compartments influence CTSD processing, as Cathepsin D has been identified in lysosomes, melanosomes, secreted fractions, and as an extracellular protein loosely bound to the matrix .

What are the best practices for using Cleaved-CTSD (G65) Antibody in Western blot experiments?

For optimal Western blot results with Cleaved-CTSD (G65) Antibody:

Sample Preparation:

• Extract proteins using lysis buffers containing protease inhibitors to prevent additional proteolysis

• Include phosphatase inhibitors if phosphorylation status may affect cleavage

• Standardize protein loading (20-50μg total protein recommended)

Western Blot Protocol:

• Separate proteins on 10-15% SDS-PAGE gels

• Transfer to PVDF or nitrocellulose membranes

• Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Incubate with Cleaved-CTSD (G65) antibody at 1:500-1:2000 dilution overnight at 4°C

• Wash thoroughly with TBST (3 × 10 minutes)

• Incubate with appropriate HRP-conjugated secondary antibody

• Develop using enhanced chemiluminescence

Validation Controls:

• Include positive controls (e.g., COS7 cells treated with etoposide 25μM for 1h)

• Perform peptide blocking experiments to confirm specificity

• Consider lysates from various cell types to assess conservation across species (effective for human and monkey samples)

Western blot analysis has demonstrated specific detection of cleaved Cathepsin D in various cell lines, with signal abolishment when blocked with the synthesized peptide .

How can Cleaved-CTSD (G65) Antibody be used in ELISA-based quantification studies?

For ELISA-based quantification of cleaved CTSD:

Sandwich ELISA Protocol:

• Coat microplate wells with capture antibody (anti-CTSD)

• Block with 1-5% BSA in PBS

• Add samples and standards

• Detect with Cleaved-CTSD (G65) antibody at 1:20000 dilution

• Add HRP-conjugated secondary antibody

• Develop with TMB substrate and measure absorbance

Standard Curve Preparation:

• Use recombinant CTSD or synthetic peptide corresponding to the cleaved region

• Prepare 2-fold serial dilutions covering the expected concentration range

• Include blank controls for background subtraction

Data Analysis:

• Generate a standard curve by plotting absorbance versus concentration

• Use four-parameter logistic regression for curve fitting

• Calculate sample concentrations based on their absorbance values

• Normalize to total protein content when comparing across different samples

This approach allows for quantitative assessment of cleaved CTSD levels across different experimental conditions or patient samples .

How can I optimize immunohistochemistry protocols using Cleaved-CTSD (G65) Antibody for tissue sections?

While the provided information does not specifically mention immunohistochemistry applications for this antibody, a methodological approach for optimization can be developed based on general principles:

Protocol Optimization Steps:

• Antigen Retrieval Optimization:

  • Test multiple antigen retrieval methods:

    • Heat-induced epitope retrieval (citrate buffer pH 6.0)

    • Enzymatic retrieval (proteinase K)

    • High pH retrieval (EDTA buffer pH 9.0)

  • Determine optimal retrieval time (10-30 minutes)

• Antibody Dilution Optimization:

  • Test serial dilutions (starting with 1:100, 1:200, 1:500)

  • Evaluate signal-to-noise ratio at each dilution

  • Optimize incubation time and temperature (overnight at 4°C vs. 1-2 hours at room temperature)

• Signal Detection System:

  • Compare different detection systems (DAB, AEC, fluorescent secondaries)

  • For fluorescence, select secondary antibodies with minimal spectral overlap with other markers

• Controls:

  • Positive tissue controls (tissues known to express cleaved CTSD)

  • Negative controls (omission of primary antibody)

  • Peptide blocking controls (pre-incubate antibody with immunizing peptide)

By methodically optimizing these parameters, researchers can develop reliable IHC protocols for studying cleaved CTSD in tissue sections.

What are common issues encountered when using Cleaved-CTSD (G65) Antibody and how can they be resolved?

Common Issues and Solutions:

Methodological Refinements:

• For challenging samples, consider enriching for lysosomal fractions where CTSD is predominantly localized

• For weak signals, implement signal enhancement systems (e.g., biotin-streptavidin amplification)

• For batch-to-batch consistency, maintain detailed records of antibody lot numbers and observed performance

How can I validate the specificity of detected signals in complex biological samples?

Validation Approaches:

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess immunizing peptide (46-95 aa region)

  • Run parallel Western blots with blocked and unblocked antibody

  • Specific signals should be eliminated or substantially reduced in the blocked condition

• Genetic Validation:

  • Use CTSD knockout or knockdown models

  • Compare signal in wild-type vs. genetically modified samples

  • Specific signals should be absent or reduced in knockdown/knockout samples

• Mass Spectrometry Validation:

  • Perform immunoprecipitation with the Cleaved-CTSD (G65) antibody

  • Analyze precipitated proteins by mass spectrometry

  • Confirm the identity of the captured proteins and cleavage sites

• Correlation with Alternative Detection Methods:

  • Use alternative antibodies recognizing different epitopes of CTSD

  • Correlation between signals from different antibodies supports specificity

These validation strategies provide multiple lines of evidence for signal specificity, enhancing confidence in experimental findings.

How does CTSD cleavage and detection differ across various experimental models and species?

Cross-Species and Model Comparisons:

The Cleaved-CTSD (G65) antibody has been validated for detecting human and monkey samples . When working with different experimental models:

Understanding these variations is crucial for accurate interpretation of experimental data across different model systems.

What are emerging applications of Cleaved-CTSD (G65) Antibody in understanding disease mechanisms?

Cleaved-CTSD (G65) antibody offers significant potential for investigating disease mechanisms beyond current applications:

• Alzheimer's Disease Research:

  • Tracking CTSD processing in relation to APP degradation and amyloid pathology

  • Investigating the relationship between CTSD cleavage and neuronal health

  • Exploring how CTSD processing affects its role in protein degradation pathways relevant to neurodegenerative diseases

• Cancer Biology:

  • Examining altered CTSD processing in various cancer types

  • Correlating cleaved CTSD levels with tumor progression and metastasis

  • Investigating CTSD processing as a potential biomarker or therapeutic target

• Lysosomal Storage Disorders:

  • Monitoring CTSD processing in neuronal ceroid lipofuscinosis and related disorders

  • Investigating how mutations affect CTSD cleavage and activation

  • Developing therapeutic strategies targeting CTSD processing pathways

These emerging applications highlight the importance of specific detection of cleaved CTSD forms in understanding pathological mechanisms.

How can complementary approaches be integrated with Cleaved-CTSD (G65) Antibody studies to gain comprehensive insights?

Integration of complementary approaches enhances the depth of Cleaved-CTSD research:

Integrated Research Strategy:

• Multi-omics Integration:

  • Combine proteomics to identify CTSD interaction partners

  • Use transcriptomics to correlate CTSD processing with gene expression patterns

  • Apply metabolomics to connect CTSD activity with metabolic changes

• Advanced Imaging Techniques:

  • Implement super-resolution microscopy to visualize CTSD processing in specific subcellular compartments

  • Use FRET-based approaches to study interactions between CTSD and substrate proteins

  • Apply live-cell imaging to track CTSD trafficking and processing in real-time

• Functional Assays:

  • Develop activity-based probes specific for cleaved CTSD

  • Correlate cleaved CTSD levels with enzymatic activity measurements

  • Design cellular assays to assess functional consequences of altered CTSD processing

• Therapeutic Development Context:

  • Screen compounds that modulate CTSD processing

  • Evaluate the impact of CTSD processing modulators on disease phenotypes

  • Develop strategies to normalize CTSD processing in pathological conditions