Cleaved-CTSC (R394) Antibody

What is CTSC and what is the significance of the R394 cleavage site?

CTSC (Cathepsin C), also known as Dipeptidyl Peptidase I, is a lysosomal cysteine proteinase that functions as a central coordinator for activating many serine proteases in immune and inflammatory cells. CTSC is expressed ubiquitously, with particularly high expression in lung, kidney, and placenta, and intermediate levels in colon, small intestine, spleen, and pancreas .

The R394 cleavage site is functionally significant because:

• It represents a critical processing point where CTSC transitions from its proenzyme form to its activated form

• Cleavage at R394 is part of the maturation process that yields the active heavy chain (HC) fragment

• This specific cleavage is essential for CTSC's ability to activate target serine proteases, including elastase, cathepsin G, and granzymes A and B

The Cleaved-CTSC (R394) Antibody specifically detects endogenous levels of the fragment of activated Cathepsin C HC protein resulting from cleavage adjacent to R394, making it a valuable tool for studying CTSC activation in various biological contexts .

What applications is the Cleaved-CTSC (R394) Antibody validated for?

Based on technical specifications, this antibody has been validated for:

The antibody is suitable for detecting cleaved CTSC in human, rat, and mouse samples, providing versatility across multiple model systems .

How should researchers prepare and store the Cleaved-CTSC (R394) Antibody?

For optimal performance and longevity:

• Storage: Store at -20°C for up to 1 year from receipt date

• Avoid repeated freeze-thaw cycles that can degrade antibody quality

• Formulation: The antibody is typically provided in liquid form in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• Concentration: Standard concentration is 1 mg/mL

• Working aliquots: For frequent use, prepare small working aliquots to avoid repeated freezing and thawing of the stock solution

How can Cleaved-CTSC (R394) Antibody be used to study protease activation cascades in immune cells?

CTSC plays a crucial role in activating multiple serine proteases involved in immune function. When studying protease activation cascades:

• Experimental design considerations:

  • Use paired antibodies against both cleaved and total CTSC to determine the activation ratio

  • Include time course analyses to track CTSC processing during immune cell activation

  • Compare CTSC cleavage patterns across different immune cell populations (neutrophils, macrophages, T cells)

• Co-detection strategy:

  • Combine Cleaved-CTSC (R394) detection with antibodies against downstream targets like granzymes

  • This approach can reveal temporal relationships between CTSC activation and subsequent protease activation

• Functional correlation:

  • Correlate cleaved CTSC levels with measurements of cellular processes like degranulation or cytotoxicity

  • Research has shown that CTSC activates serine proteases such as elastase, cathepsin G, and granzymes A and B, which are critical for immune cell function

• Stimulation protocols:

  • Use physiologically relevant stimuli (e.g., pathogen-associated molecular patterns, inflammatory cytokines)

  • Document changes in cleaved CTSC levels during immune cell maturation and activation

What are the best validation methods for confirming Cleaved-CTSC (R394) Antibody specificity in experimental systems?

To ensure reliable results, researchers should employ multiple validation approaches:

• Genetic validation:

  • Use CTSC knockout cells/tissues as negative controls

  • Employ CRISPR-edited cell lines with mutations at the R394 cleavage site

  • Compare with cells expressing cleavage-resistant CTSC mutants

• Biochemical validation:

  • Perform peptide competition assays using the immunizing peptide (amino acids 345-394 of human CTSC)

  • Conduct immunoprecipitation followed by mass spectrometry to confirm the identity of the detected protein

  • Compare detection patterns with other validated anti-CTSC antibodies targeting different regions

• Functional validation:

  • Correlate antibody signals with enzymatic activity assays specific for CTSC

  • Use protease inhibitors to block CTSC processing and confirm the disappearance of the cleaved form

  • Stimulate cells with known inducers of CTSC activation and verify increased detection

• Technical controls:

  • Include secondary antibody-only controls to rule out non-specific binding

  • Use recombinant cleaved CTSC as a positive control

  • Perform western blots under reducing and non-reducing conditions to confirm specificity

How does CTSC cleavage compare in different disease models, and how can the Cleaved-CTSC (R394) Antibody help elucidate these differences?

CTSC cleavage patterns vary across disease contexts, offering insights into pathophysiological mechanisms:

• Cancer research applications:

  • Studies indicate alterations in protease networks in various cancer types

  • The R394 antibody can help track CTSC activation in tumor microenvironments

  • Research suggests connections between CTSC and immune cell functions that may influence anti-tumor responses

• Inflammatory disease models:

  • CTSC activates neutrophil-derived serine proteases implicated in inflammatory conditions

  • The antibody can help quantify CTSC activation status in affected tissues

  • Compare cleaved CTSC levels between acute and chronic inflammation models

• Infectious disease research:

  • CTSC-activated proteases play roles in host defense against pathogens

  • The antibody can track CTSC activation during infection progression

  • Studies suggest CTSC-activated proteases may influence pathogen clearance mechanisms

• Methodological considerations:

  • Use standardized sampling and processing procedures across disease models

  • Employ multiple tissue preparation techniques to account for potential artifacts

  • Consider timing of sample collection relative to disease progression

What methodological approaches can help resolve discrepancies in Cleaved-CTSC detection across different experimental systems?

When facing inconsistent results:

• Sample preparation optimization:

  • Test different lysis buffers with varying protease inhibitor compositions

  • Standardize sample processing time to minimize ex vivo proteolysis

  • Optimize protein extraction methods for different tissue types

  • Consider rapid freezing techniques to preserve in vivo cleavage status

• Antibody validation across systems:

  • Verify antibody performance in each experimental system using known controls

  • Determine optimal antibody concentration for each application and sample type

  • Test multiple detection systems (chemiluminescence, fluorescence) for optimal signal-to-noise ratio

• Reproducibility enhancement:

  • Standardize protocols across laboratory members

  • Document lot-to-lot variation of the antibody

  • Incorporate quantitative standards for normalization across experiments

• Complementary approach integration:

  • Use multiple antibodies targeting different regions of cleaved CTSC

  • Complement antibody-based detection with activity-based probes

  • Consider using parallel techniques like mass spectrometry to confirm cleavage events

What is the optimal protocol for using Cleaved-CTSC (R394) Antibody in Western blotting?

For optimal Western blot results:

• Sample preparation:

  • Use fresh tissue/cell lysates whenever possible

  • Include protease inhibitor cocktail (excluding cysteine protease inhibitors if studying active CTSC)

  • Standardize protein loading (20-50 μg total protein per lane)

  • Prepare samples in reducing buffer containing β-mercaptoethanol

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal resolution

  • Consider gradient gels (4-15%) when analyzing complex samples

  • Transfer to PVDF membrane (recommended over nitrocellulose for cleaved proteins)

  • Verify transfer efficiency with reversible protein stain

• Antibody incubation:

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

  • Dilute primary antibody 1:500-1:2000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

  • Wash 4x for 5 minutes each with TBST

• Detection optimization:

  • Use HRP-conjugated secondary antibody at 1:5000-1:10000 dilution

  • Consider enhanced sensitivity detection reagents for low abundance targets

  • Optimize exposure time to avoid signal saturation

  • Include molecular weight markers to confirm band size (expected MW for cleaved CTSC may vary based on glycosylation status)

How can researchers optimize the use of Cleaved-CTSC (R394) Antibody in immunohistochemistry applications?

While specific IHC validation may not be explicitly mentioned in the provided data, researchers can apply these general principles:

• Tissue preparation considerations:

  • Test both formalin-fixed paraffin-embedded (FFPE) and frozen sections

  • Optimize antigen retrieval methods (citrate buffer pH 6.0 is often a good starting point)

  • Consider testing different fixation protocols to preserve epitope integrity

• Staining protocol optimization:

  • Start with antibody dilution of 1:100-1:200 and optimize based on results

  • Extend primary antibody incubation time (overnight at 4°C may improve signal)

  • Include blocking steps for endogenous peroxidase and biotin if applicable

  • Consider signal amplification systems for low abundance targets

• Controls and validation:

  • Include tissue known to express high levels of cleaved CTSC (lung, kidney, placenta)

  • Use CTSC knockout tissue as negative control when available

  • Perform peptide competition controls using the immunizing peptide

• Co-localization studies:

  • Consider double-staining with cell type-specific markers to identify cleaved CTSC-positive cell populations

  • Use sequential staining protocols to minimize cross-reactivity

  • Employ spectral unmixing if using fluorescent detection systems

What approaches can researchers use to quantify cleaved CTSC levels in experimental samples?

For accurate quantification:

• Western blot densitometry:

  • Use validated quantification software (ImageJ, Image Lab, etc.)

  • Normalize cleaved CTSC signal to appropriate loading controls

  • Generate standard curves using recombinant protein when possible

  • Present data as ratio of cleaved to total CTSC to account for expression variations

• ELISA-based quantification:

  • The antibody has been validated for ELISA at 1:20000 dilution

  • Develop sandwich ELISA using capture antibody against total CTSC and detection with Cleaved-CTSC (R394) Antibody

  • Include standard curves with known concentrations of recombinant protein

  • Consider developing a high-sensitivity ELISA for low abundance samples

• Image-based quantification:

  • For immunohistochemistry/immunofluorescence, use automated image analysis

  • Establish consistent thresholding parameters across experimental groups

  • Quantify both signal intensity and percentage of positive cells

  • Consider 3D reconstruction for volumetric quantification in tissue sections

• Flow cytometry applications:

  • Optimize fixation and permeabilization conditions for intracellular staining

  • Use median fluorescence intensity (MFI) for quantitative comparisons

  • Include fluorescence-minus-one (FMO) controls

  • Consider phospho-flow protocols for detecting dynamic changes in CTSC cleavage

How does post-translational modification of CTSC affect detection with the Cleaved-CTSC (R394) Antibody?

CTSC undergoes several post-translational modifications that may influence antibody detection:

• N-glycosylation considerations:

  • CTSC is N-glycosylated at multiple sites (Asn-29, Asn-53, Asn-119, Asn-276)

  • Different glycosylation patterns can affect apparent molecular weight on Western blots

  • Consider using deglycosylation enzymes (PNGase F) to confirm band identity in complex samples

  • N-glycosylation at Asn-29 is mediated by STT3B-containing complexes, while glycosylation at Asn-53, Asn-119, and Asn-276 is mediated by STT3A-containing complexes

• Disulfide bonding effects:

  • CTSC contains disulfide bonds that maintain its structure

  • Running samples under non-reducing conditions may preserve epitopes dependent on tertiary structure

  • The exclusion domain of CTSC is held together by disulfide bonds that may influence antibody accessibility

• Proteolytic processing:

  • CTSC undergoes multiple cleavage events during maturation

  • The R394 cleavage is one of several processing steps

  • Consider using multiple antibodies targeting different cleaved forms for comprehensive analysis

  • Approximately 50% of CTSC complexes undergo cleavage at position 58 or 61 in the exclusion domain

• Experimental recommendations:

  • Include controls treated with specific inhibitors of post-translational modifications

  • Consider subcellular fractionation to distinguish different pools of cleaved CTSC

  • Verify results with complementary approaches like mass spectrometry

What are the key considerations when using Cleaved-CTSC (R394) Antibody alongside other antibodies in multiplex detection systems?

For successful multiplex applications:

• Antibody compatibility assessment:

  • Verify primary antibody host species compatibility to avoid cross-reactivity

  • Test each antibody individually before combining in multiplex system

  • Consider using directly conjugated primary antibodies to reduce background

• Signal separation strategies:

  • Use primary antibodies from different host species

  • Select fluorophores with minimal spectral overlap for immunofluorescence

  • Consider sequential detection protocols for challenging combinations

  • Use tyramide signal amplification for low abundance targets

• Technical considerations for co-detection:

  • Optimize fixation and permeabilization protocols that work for all target epitopes

  • Test different antigen retrieval methods if targets respond differently

  • Increase blocking time and stringency to reduce non-specific binding

  • Include appropriate compensation controls for fluorescence-based detection

• Data analysis approaches:

  • Use co-localization analysis tools for spatial relationships

  • Employ advanced image analysis software for quantitative co-expression studies

  • Consider single-cell analysis approaches for heterogeneous samples