Cleaved-CTSG (I21) Antibody

What is Cleaved-CTSG (I21) Antibody and what epitope does it specifically recognize?

Cleaved-CTSG (I21) Antibody is a polyclonal antibody that specifically recognizes the N-terminal region of Cathepsin G (CTSG), particularly detecting endogenous levels of fragments resulting from proteolytic cleavage adjacent to the Isoleucine at position 21 (Ile21) . This antibody was generated using a synthesized peptide derived from the human Cathepsin G sequence and has been affinity-purified from rabbit antiserum using epitope-specific immunogen chromatography . Unlike antibodies that recognize intact proteins regardless of their activation state, this antibody specifically detects the cleaved form, making it valuable for studying protease activation and processing events in biological systems .

How does the specificity of Cleaved-CTSG (I21) Antibody compare to other antibodies targeting Cathepsin G?

The Cleaved-CTSG (I21) Antibody differs from standard anti-Cathepsin G antibodies in its high specificity for the cleaved form adjacent to Ile21 . While conventional antibodies typically recognize epitopes present on both inactive and active forms of the enzyme, the Cleaved-CTSG (I21) Antibody selectively binds to the neo-epitope exposed only after proteolytic processing . This specificity is similar in principle to the mechanism observed with antibodies like mAb 2095-2, which targets cleaved IgG molecules but not their intact counterparts . For researchers investigating activation-dependent processes, this cleaved-form specificity provides a significant advantage by enabling selective detection of the processed enzyme without interference from the inactive precursor forms present in biological samples.

What are the key technical specifications of the Cleaved-CTSG (I21) Antibody that researchers should be aware of?

The Cleaved-CTSG (I21) Antibody has several important technical specifications that researchers should consider when designing experiments:

This comprehensive characterization allows researchers to make informed decisions about experimental design and technical implementation when using this antibody .

How can Cleaved-CTSG (I21) Antibody be utilized to study neutrophil activation and inflammatory responses?

Cleaved-CTSG (I21) Antibody offers a powerful tool for investigating neutrophil activation and inflammatory responses, as Cathepsin G is predominantly expressed in neutrophils and plays critical roles in immune defense and inflammatory processes. To study neutrophil activation, researchers can design experiments that compare the levels of cleaved CTSG (using this antibody) versus total CTSG (using an antibody that recognizes both forms) under various stimulation conditions. This approach provides insight into the proportion of activated enzyme present in different physiological or pathological states.

For inflammatory response studies, researchers can use the antibody in immunohistochemistry or immunofluorescence experiments to track the spatial and temporal patterns of CTSG activation in tissues experiencing inflammation. Additionally, the antibody can be employed in time-course experiments examining neutrophil extracellular trap (NET) formation, where CTSG is known to be an important component. By specifically detecting the cleaved, active form of CTSG, researchers can more precisely correlate enzyme activation with functional outcomes in inflammatory cascades, potentially revealing new therapeutic targets or biomarkers for inflammatory conditions.

What considerations should be made when using Cleaved-CTSG (I21) Antibody for detecting post-translational modifications?

When using Cleaved-CTSG (I21) Antibody to detect post-translational modifications (PTMs), several important considerations should be addressed. First, sample preparation is crucial—proteolytic processing is sensitive to experimental conditions, so researchers must carefully control factors like temperature, pH, and exposure to proteases during sample handling to avoid artificial cleavage or degradation. Use of appropriate protease inhibitors is essential, but researchers must be mindful that these inhibitors should not interfere with the natural cleavage event at Ile21 that generates the epitope.

Second, validation experiments should include positive controls (samples known to contain cleaved CTSG) and negative controls (samples where CTSG cleavage is inhibited). Additionally, researchers should consider cellular compartmentalization when interpreting results, as CTSG can be found in various locations including azurophilic granules, phagolysosomes, and extracellular space following degranulation.

Finally, for comprehensive PTM analysis, complementary techniques should be employed alongside antibody-based detection. These might include mass spectrometry to precisely identify cleavage sites and additional modifications, or activity-based assays to correlate cleaved CTSG detection with functional enzyme activity. This multi-faceted approach provides the most robust analysis of CTSG processing and activation in biological systems.

What is the optimal storage protocol for maintaining Cleaved-CTSG (I21) Antibody activity, and how can researchers assess potential activity loss?

To assess potential activity loss, researchers should implement regular quality control measures. A simple approach is to include a well-characterized positive control sample in each experiment and monitor signal intensity over time. More systematically, researchers can prepare a standard curve using serial dilutions of a positive control lysate and compare antibody performance across multiple experiments. Any significant decrease in signal strength or increase in non-specific background may indicate antibody deterioration. Additionally, maintain a laboratory record documenting the number of freeze-thaw cycles and storage duration for each aliquot to correlate with any observed performance changes. If diminished activity is suspected, compare results with a fresh aliquot to determine whether the antibody or other experimental factors are responsible for the changes.

How can researchers optimize Western blot protocols specifically for Cleaved-CTSG (I21) Antibody detection?

Optimizing Western blot protocols for Cleaved-CTSG (I21) Antibody requires attention to several key parameters. First, sample preparation is critical—use a lysis buffer containing appropriate protease inhibitors to prevent artifactual cleavage, but ensure these inhibitors won't affect the natural Ile21 cleavage site. Consider using both reducing and non-reducing conditions in parallel experiments, as the epitope recognition might be affected by disulfide bond reduction.

For the gel electrophoresis step, 12-15% polyacrylamide gels are generally recommended for optimal resolution of Cathepsin G fragments (approximately 28-30 kDa for the mature protein). During transfer, PVDF membranes may provide better protein retention than nitrocellulose for this particular application. For blocking, 5% non-fat dry milk in TBST is typically effective, but if background issues occur, consider BSA as an alternative blocking agent.

The antibody application should follow the manufacturer's recommended dilution range (1:500-1:2000) , starting with a mid-range dilution (1:1000) and adjusting based on results. Extended primary antibody incubation (overnight at 4°C) often yields better results than shorter incubations at room temperature. For detection, enhanced chemiluminescence (ECL) systems generally provide good sensitivity, but for weaker signals, consider using amplified detection systems.

Additionally, when analyzing results, remember that the antibody specifically detects the cleaved form, so band patterns will differ from those seen with antibodies that recognize total Cathepsin G. A549 cells have been validated as a positive control for this antibody, as shown in Western blot analysis .

What strategies can be employed to reduce non-specific binding when using Cleaved-CTSG (I21) Antibody in immunohistochemistry?

To reduce non-specific binding when using Cleaved-CTSG (I21) Antibody in immunohistochemistry (IHC), researchers should implement a comprehensive optimization strategy. Begin with proper tissue fixation and processing, as overfixation can mask epitopes while underfixation may compromise tissue morphology. For formalin-fixed, paraffin-embedded tissues, effective antigen retrieval is essential—test both heat-mediated (citrate buffer, pH 6.0) and enzymatic methods to determine optimal epitope exposure.

Blocking steps are particularly important for polyclonal antibodies like Cleaved-CTSG (I21). Use a combination approach with 5-10% normal serum from the same species as the secondary antibody, plus 1-3% BSA to block both Fc receptors and non-specific protein interactions. Consider adding 0.1-0.3% Triton X-100 for better antibody penetration, particularly in tissues with high neutrophil content where CTSG is abundant.

Further optimization can be achieved through antibody dilution testing (starting at 1:500 and adjusting based on results) and incubation conditions (overnight at 4°C often reduces background compared to shorter incubations at room temperature). Additionally, include appropriate negative controls: (1) omission of primary antibody, (2) isotype-matched irrelevant antibody, and (3) pre-adsorption of the antibody with the immunizing peptide when available.

For tissues with high endogenous peroxidase activity (like neutrophil-rich samples), extended hydrogen peroxide quenching (3% H₂O₂ for 15-20 minutes) before antibody incubation can significantly reduce background. Finally, consider using tyramide signal amplification systems for weak signals rather than simply increasing antibody concentration, as higher concentrations often increase non-specific binding proportionally with specific signals.

How can Cleaved-CTSG (I21) Antibody be integrated with other methodologies to study protease networks in inflammatory diseases?

Integrating Cleaved-CTSG (I21) Antibody with complementary methodologies creates powerful approaches for deciphering protease networks in inflammatory diseases. One sophisticated strategy involves combining immunodetection of cleaved CTSG with activity-based protein profiling (ABPP). This approach uses biotinylated activity-based probes that covalently bind to active proteases, followed by pull-down and parallel analysis with Cleaved-CTSG (I21) Antibody detection. This combination distinguishes between cleaved-but-inactive and fully active forms of the enzyme, providing deeper insights into post-translational regulation.

Another powerful integration involves coupling Cleaved-CTSG (I21) Antibody immunoprecipitation with mass spectrometry (IP-MS). This technique allows identification of protein complexes and substrates specifically associated with the activated form of CTSG, potentially revealing novel targets in inflammatory cascades. For spatiotemporal analysis in tissues, multiplexed immunofluorescence combining Cleaved-CTSG (I21) Antibody with antibodies against other proteases (e.g., neutrophil elastase, proteinase 3) and their substrates enables visualization of coordinated protease activation networks.

In functional studies, researchers can correlate cleaved CTSG levels (detected via this antibody) with real-time substrate cleavage assays using fluorogenic peptides specific for different proteases. This correlation between enzyme activation state and catalytic activity provides mechanistic insights into protease regulation. Additionally, integrating these approaches with single-cell technologies like CyTOF or single-cell RNA-seq creates unprecedented opportunities to link CTSG activation states with cell-specific transcriptional programs in heterogeneous inflammatory environments.

What experimental approaches can differentiate between functionally active Cleaved-CTSG and inactive forms in complex biological samples?

Differentiating between functionally active cleaved CTSG and inactive forms requires sophisticated experimental approaches beyond simple detection. One effective strategy employs a dual-detection system combining the Cleaved-CTSG (I21) Antibody with activity-based probes. Researchers can use serine protease-specific activity-based probes like fluorophosphonates (FP) or diphenyl phosphonates, which covalently bind only to catalytically active enzymes. By comparing the population detected by Cleaved-CTSG (I21) Antibody (all cleaved forms) with those bound by activity probes (only catalytically active forms), researchers can quantify the proportion of cleaved-but-inactive enzymes.

Another advanced approach involves immunocapture followed by activity assays. The Cleaved-CTSG (I21) Antibody can be used to immunoprecipitate all cleaved forms from biological samples, followed by incubation with fluorogenic or chromogenic CTSG-specific substrates like Suc-Ala-Ala-Pro-Phe-pNA. The measured enzymatic activity normalized to the amount of immunoprecipitated protein provides a direct assessment of the specific activity of the cleaved population.

For in situ analysis in tissues or cells, researchers can employ proximity ligation assays (PLA) that combine the Cleaved-CTSG (I21) Antibody with antibodies against known CTSG inhibitors like serpins. Strong PLA signals would indicate cleaved-but-inhibited (inactive) enzyme, while cleaved active enzyme would show minimal PLA signal with inhibitors. This approach provides spatial information about the activation state within tissues.

Additionally, native gel electrophoresis combined with Western blotting can separate different conformational states of cleaved CTSG based on their interaction with inhibitors, providing a biochemical means to distinguish active from inactive cleaved forms in complex samples.

How can researchers design studies to investigate the relationship between CTSG cleavage and its role in neutrophil extracellular trap (NET) formation?

Designing robust studies to investigate the relationship between CTSG cleavage and NET formation requires multifaceted experimental approaches. Begin with time-course experiments using neutrophils stimulated with known NET inducers (PMA, LPS, or calcium ionophores) and collect samples at defined intervals (15, 30, 60, 120, 180 minutes). At each timepoint, process parallel samples for (1) Western blotting with Cleaved-CTSG (I21) Antibody to quantify cleavage kinetics, (2) fluorescence microscopy to visualize NET formation, and (3) CTSG activity assays using specific substrates. This temporal correlation between CTSG cleavage, activity, and NET formation provides mechanistic insights into the sequential events.

To establish causality rather than correlation, develop intervention studies using selective CTSG inhibitors or genetic approaches (CRISPR/Cas9 or siRNA in cell lines, or neutrophils from CTSG knockout mice). Compare how inhibiting CTSG activation affects NET formation across different stimuli, as stimulus-specific pathways may have different dependencies on CTSG activity. Additionally, design rescue experiments where recombinant pre-cleaved active CTSG is introduced into inhibitor-treated or CTSG-deficient neutrophils to determine if this can restore NET formation capability.

For spatial analysis, implement super-resolution microscopy (STORM, PALM, or SIM) using the Cleaved-CTSG (I21) Antibody alongside markers for chromatin (Hoechst), neutrophil elastase, and citrullinated histones. This approach can reveal the spatiotemporal dynamics of CTSG activation relative to chromatin decondensation and other molecular events in NET formation. Additionally, examine how pharmacological manipulation of upstream pathways (e.g., NADPH oxidase inhibition, PAD4 inhibition) affects CTSG cleavage to position it within the known NET formation cascade.

Finally, design translational studies comparing the kinetics and extent of CTSG cleavage in neutrophils from healthy donors versus patients with diseases characterized by aberrant NET formation (like lupus, rheumatoid arthritis, or sepsis). Correlating disease phenotypes with patterns of CTSG activation may reveal pathologically relevant mechanisms and potential therapeutic targets.

What are the most common causes of false-positive or false-negative results when using Cleaved-CTSG (I21) Antibody, and how can they be mitigated?

When working with Cleaved-CTSG (I21) Antibody, several factors can lead to misleading results. For false-positives, the most common causes include: (1) Artifactual cleavage during sample preparation—neutrophil-rich samples contain numerous proteases that can generate cleavage products if inadequately inhibited; (2) Cross-reactivity with structurally similar proteases, particularly in overexpression systems; and (3) Non-specific binding to denatured proteins exposed during harsh fixation procedures in immunohistochemistry.

To mitigate these false-positives, implement stringent sample preparation protocols with comprehensive protease inhibitor cocktails, but avoid serine protease inhibitors when studying natural CTSG cleavage. Include negative controls like CTSG-knockout samples or tissues from CTSG-deficient mice. For immunohistochemistry, optimize fixation procedures and antibody concentrations, and consider antigen retrieval optimization to reduce non-specific binding while enhancing specific signals.

False-negatives frequently result from: (1) Epitope masking due to protein-protein interactions or post-translational modifications near the cleavage site; (2) Inadequate sample preparation leading to protein degradation or epitope destruction; and (3) Suboptimal antibody concentration or incubation conditions. To address these issues, test multiple sample preparation methods, including different lysis buffers and denaturation conditions. Consider multiple antigen retrieval methods for fixed tissues, and test a range of antibody concentrations beyond the manufacturer's recommendations. Additionally, include positive control samples (e.g., A549 cells ) in every experiment to validate assay functionality.

For both false-positive and false-negative concerns, validation with complementary techniques is essential. Consider correlating antibody-based detection with mass spectrometry identification of cleavage products or activity-based assays that measure functional enzyme.

How should researchers interpret complex banding patterns in Western blots using Cleaved-CTSG (I21) Antibody?

Higher molecular weight bands (40-50 kDa) may indicate CTSG complexed with small inhibitors or post-translationally modified forms (glycosylated, ubiquitinated). Bands around 35-37 kDa could represent pro-CTSG that has undergone partial processing. Lower molecular weight bands (15-25 kDa) often represent additional proteolytic fragments resulting from secondary cleavage events beyond the initial activation cleavage.

To systematically interpret these patterns, researchers should:

• Run size markers and positive control samples (like A549 cells ) alongside experimental samples

• Compare reducing vs. non-reducing conditions to identify bands affected by disulfide bonds

• Perform peptide competition assays using the immunizing peptide to identify specific vs. non-specific bands

• Conduct parallel Western blots with antibodies recognizing different CTSG epitopes to create a more complete picture of processing events

• Consider sample pretreatment with glycosidases or phosphatases to identify bands resulting from these modifications

What approaches can resolve contradictory results between Cleaved-CTSG (I21) Antibody detection and functional assays of Cathepsin G activity?

Contradictions between Cleaved-CTSG (I21) Antibody detection and functional assays of Cathepsin G activity are not uncommon and often provide important biological insights rather than simply experimental artifacts. To resolve such discrepancies, several methodological approaches can be employed.

First, determine whether the contradiction reflects biological reality—cleaved CTSG might be present but enzymatically inactive due to endogenous inhibitors like serpins. To test this hypothesis, perform size exclusion chromatography or native gel electrophoresis to separate free CTSG from inhibitor-bound complexes, followed by parallel analysis with both the antibody and activity assays. Additionally, treat samples with chaotropic agents that disrupt protein-protein interactions without denaturing the enzyme to potentially release CTSG from inhibitor complexes, then reassess activity.

Second, examine post-translational modifications that might affect activity but not antibody recognition. Phosphorylation, oxidation, or glycosylation could impact the catalytic activity without affecting the cleaved epitope recognized by the antibody. Mass spectrometry analysis of immunoprecipitated CTSG can identify such modifications.

Third, consider microenvironmental factors affecting enzyme activity but not antibody binding. Factors like pH, ionic strength, or specific cofactors might be critical for activity in functional assays but irrelevant for antibody detection. Systematically alter these conditions in activity assays to determine if they explain the discrepancy.

Finally, implement advanced approaches like proximity-dependent biotin identification (BioID) or enzyme-substrate crosslinking to identify proteins interacting with cleaved CTSG in situ, potentially explaining activity regulation. This comprehensive troubleshooting not only resolves contradictions but often leads to novel insights into CTSG regulation in complex biological systems.

How might Cleaved-CTSG (I21) Antibody be utilized in single-cell analysis platforms to study heterogeneity in neutrophil activation states?

The application of Cleaved-CTSG (I21) Antibody in single-cell analysis platforms offers unprecedented opportunities to characterize heterogeneity in neutrophil activation states. In mass cytometry (CyTOF) platforms, the antibody can be metal-conjugated (e.g., with isotopically pure lanthanides) and combined with surface markers and other intracellular activation indicators to create high-dimensional profiles of individual neutrophils. This approach would reveal distinct neutrophil subpopulations based on CTSG activation status correlated with other functional markers.

For imaging-based single-cell analysis, multiplexed immunofluorescence using iterative staining and bleaching techniques like CODEX or Cyclic Immunofluorescence (CyCIF) can incorporate the Cleaved-CTSG (I21) Antibody alongside dozens of other markers. This would provide spatial information about CTSG activation within individual cells and in relation to neighboring cells in the tissue microenvironment. These technologies are particularly valuable for studying neutrophil heterogeneity in complex inflammatory tissues where diverse activation states coexist.

Single-cell RNA-sequencing approaches can be complemented with protein analysis using technologies like CITE-seq, where oligonucleotide-tagged Cleaved-CTSG (I21) Antibody would allow simultaneous detection of transcriptome and cleaved CTSG protein level in the same cells. This could reveal relationships between transcriptional programs and post-translational activation of CTSG, potentially identifying regulatory mechanisms of neutrophil functional diversity.

Additionally, microfluidic platforms enabling single-cell proteomics could incorporate the antibody for analyzing CTSG activation in conjunction with other protease activations at single-cell resolution. This would provide insights into the coordination of protease networks at the individual cell level, potentially identifying "leader" and "follower" cells in neutrophil activation cascades.

What insights might comparative studies using both anti-total CTSG and Cleaved-CTSG (I21) antibodies provide about proteolytic regulation across different disease models?

Comparative studies utilizing both anti-total CTSG and Cleaved-CTSG (I21) antibodies across disease models can provide multidimensional insights into proteolytic regulation. Such dual-antibody approaches enable calculation of the "activation ratio" (cleaved/total CTSG) as a quantitative measure of CTSG activation state. This ratio may serve as a more informative biomarker than absolute levels of either form alone, potentially revealing disease-specific patterns of dysregulation.

In inflammatory disorders like rheumatoid arthritis, COPD, or inflammatory bowel disease, measuring this activation ratio in tissue biopsies, bronchoalveolar lavage fluid, or mucosal samples could identify disease subtypes characterized by differential protease activation rather than just protease abundance. Similarly, in infectious disease models, tracking changes in the activation ratio during infection progression might reveal pathogen-specific strategies for manipulating host proteolytic networks.

For cancer studies, dual antibody approaches could distinguish between tumors with high CTSG expression versus those with enhanced CTSG activation, potentially correlating with different invasion or metastasis mechanisms. This distinction might have significant implications for therapeutic strategies targeting neutrophil functions in the tumor microenvironment.

In longitudinal studies of acute conditions like sepsis or acute respiratory distress syndrome, monitoring the activation ratio over time could identify critical transition points in disease progression before clinical parameters change, potentially guiding early intervention. Additionally, examining how the ratio responds to various therapies could provide mechanistic insights into treatment effects on neutrophil biology.

Finally, for fundamental neutrophil biology, combined immunofluorescence with both antibodies can create spatial maps of CTSG processing within neutrophil compartments, potentially identifying specific granule subsets or microdomains where activation preferentially occurs. This approach might reveal previously unrecognized heterogeneity in neutrophil granule composition and activation dynamics.

How can emerging technologies in functional proteomics be combined with Cleaved-CTSG (I21) Antibody to study context-dependent roles of neutrophil proteases?

Emerging functional proteomics technologies can be powerfully combined with Cleaved-CTSG (I21) Antibody to unravel the context-dependent roles of neutrophil proteases. One cutting-edge approach integrates this antibody with proximity-dependent labeling methods like TurboID or APEX2. By genetically fusing these proximity labeling enzymes to CTSG-interacting proteins and using the Cleaved-CTSG (I21) Antibody for co-localization studies, researchers can identify proteins that specifically interact with the activated form of CTSG in living cells under various physiological conditions.

Another innovative integration involves combining the antibody with advanced proteomics techniques like Terminal Amine Isotopic Labeling of Substrates (TAILS) or Proteomic Identification of protease Cleavage Sites (PICS). In these approaches, researchers can compare the degradome profiles generated by neutrophils in different activation states (quantified using the Cleaved-CTSG (I21) Antibody) to correlate specific substrate processing events with defined levels of CTSG activation. This creates a functional map linking enzyme activation to biological outcomes.

Microfluidic systems represent another frontier, allowing researchers to construct artificial tissue niches where neutrophils can be challenged with defined stimuli while monitoring CTSG activation in real-time using fluorescently-labeled Cleaved-CTSG (I21) Antibody. By simultaneously tracking neutrophil behavior (migration, phagocytosis, NET formation) alongside CTSG activation, researchers can establish direct causal relationships between enzyme activation and functional outcomes.

For in vivo studies, intravital microscopy combined with fluorescently conjugated Cleaved-CTSG (I21) Antibody (either directly injected or expressed as an intrabody) can visualize CTSG activation during neutrophil recruitment to sites of inflammation or infection. This approach reveals the spatiotemporal dynamics of protease activation in living tissues, potentially identifying tissue-specific regulatory mechanisms.

Finally, computational proteomics approaches can integrate data from these various platforms with structural biology information to model how CTSG activation affects substrate selection and processing efficiency in different microenvironments, creating predictive frameworks for understanding neutrophil protease functions across diverse physiological and pathological contexts.