Cleaved-CTSA (R326) Antibody

What is Cleaved-CTSA (R326) Antibody and what epitope does it recognize?

Cleaved-CTSA (R326) Antibody is a research-grade antibody that specifically recognizes the activated 32 kDa fragment of Cathepsin A (CTSA) protein resulting from proteolytic cleavage adjacent to arginine 326. The antibody targets the immunogen region spanning amino acids 277-326 of human PPGB (Protective Protein/Cathepsin A) . It is available in both polyclonal and monoclonal forms, with polyclonal versions typically produced in rabbit and monoclonal versions in mouse .

What are the main applications for Cleaved-CTSA (R326) Antibody?

This antibody has been validated primarily for Western Blot (WB) and ELISA applications. For Western Blot, the recommended dilution range is 1:500-1:2000, while for ELISA, the optimal dilution is approximately 1:10000 . The antibody has not been extensively validated for other applications such as immunohistochemistry or immunofluorescence, though these potential applications may be explored by researchers with appropriate optimization .

What species reactivity has been confirmed for this antibody?

Cleaved-CTSA (R326) Antibody has been confirmed to react with both human and mouse samples . This cross-reactivity makes it valuable for comparative studies across these species. It should be noted that the specific amino acid positions may differ between species (e.g., R326 in human corresponds to R314 in mouse) , but the antibody recognizes the homologous region in both species.

What is the recommended storage protocol for Cleaved-CTSA (R326) Antibody?

For optimal performance and longevity, store the antibody at -20°C for up to one year from the date of receipt . It's crucial to avoid repeated freeze-thaw cycles as these can degrade antibody performance . For longer-term storage, aliquoting the antibody before freezing is recommended to minimize freeze-thaw cycles. The antibody is typically supplied in a stabilizing buffer containing PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide .

How should I optimize Western Blot protocols when using Cleaved-CTSA (R326) Antibody?

When optimizing Western Blot protocols with Cleaved-CTSA (R326) Antibody, begin with a mid-range dilution (1:1000) and adjust based on signal strength. For sample preparation, ensure complete protein denaturation by boiling samples in SDS sample buffer containing a reducing agent like DTT . Use 4-20% gradient gels for optimal separation of the 32 kDa fragment. During transfer, PVDF membranes often yield better results than nitrocellulose for this particular antibody. Block with 5% skim milk in TBS for 45 minutes at room temperature before antibody incubation . For detection, both chemiluminescence and fluorescence-based methods are compatible, though chemiluminescence may provide better sensitivity for detecting the cleaved form specifically .

What are the key considerations for developing a competitive ELISA using Cleaved-CTSA (R326) Antibody?

For competitive ELISA development using this antibody, consider the following methodology: Coat 96-well plates with purified CTSA protein (0.5 μg/well) overnight at 4°C . After blocking with 2% PBS-BSA for 1 hour, prepare an antibody cocktail containing Cleaved-CTSA (R326) Antibody at a constant concentration (40 μg/mL) and the competing antibody or sample at increasing concentrations (0-80 μg/mL) . The decrease in binding can be detected using appropriate secondary antibodies. For quantitative analysis, plot the competitive binding curve using software like GraphPad Prism . This approach is particularly valuable for epitope mapping or for measuring the concentration of cleaved CTSA in experimental samples.

How can I validate the specificity of Cleaved-CTSA (R326) Antibody in my experimental system?

Validating antibody specificity is crucial for reliable research outcomes. For Cleaved-CTSA (R326) Antibody, implement a multi-faceted validation approach: First, include both positive controls (samples known to contain cleaved CTSA) and negative controls (CTSA-knockout cells or tissues) . Second, perform peptide competition assays using the immunizing peptide (aa 277-326) to confirm binding specificity . Third, compare staining patterns with alternative antibodies targeting different CTSA epitopes. Fourth, verify the molecular weight of the detected band (32 kDa) corresponds to the expected cleaved fragment. Finally, if possible, generate cleaved CTSA through controlled proteolysis and confirm detection by the antibody increases proportionally with cleavage .

How can Cleaved-CTSA (R326) Antibody be used to study protease-resistant antibody engineering?

Cleaved-CTSA (R326) Antibody provides a valuable tool for studying protease-resistant antibody engineering. Researchers can utilize this antibody to detect specific cleavage events in the lower hinge region of IgG1, which results in loss of Fc-mediated cell-killing functions without compromising antigen binding capability . In experimental designs, compare wild-type antibodies with engineered protease-resistant variants by subjecting them to CTSA or other proteases, then analyze the cleavage patterns using Western blot with anti-cleaved detection . This approach allows quantitative assessment of protease resistance conferred by specific mutations incorporated into the CH2 region of engineered monoclonal antibodies. The methodology has been successfully employed to demonstrate that certain mutations not only restore functional activities to cleaved IgG1 but can substantially enhance Fc effector functions .

What strategies can be employed to use Cleaved-CTSA (R326) Antibody in multiplex assays with other antibodies?

For multiplex assay development incorporating Cleaved-CTSA (R326) Antibody, several strategies can be implemented: First, develop a multiplex competition assay based on electro-chemiluminescence immunoassay (ECLIA) platforms, which offer high sensitivity with low variability and a wide linear range over 4-5 logs . When combining with other antibodies, carefully select those raised in different host species or use isotype-specific secondary antibodies to avoid cross-reactivity. For multiplex Western blots, use fluorescently-labeled secondary antibodies with distinct emission spectra. In flow cytometry applications, pair with antibodies conjugated to non-overlapping fluorophores . When designing competition-based multiplex assays, test for epitope overlap by examining whether binding of one antibody affects binding of others . This approach has been successfully used in developing assays like CBASQE (Competition-Based Assay for Serological Quantification and Equivalency) .

How does Cleaved-CTSA (R326) Antibody performance compare between polyclonal and monoclonal formats for detecting low abundance targets?

The performance comparison between polyclonal and monoclonal formats of Cleaved-CTSA (R326) Antibody reveals important considerations for detecting low abundance targets. Polyclonal antibodies (such as those raised in rabbit) generally provide higher sensitivity due to their ability to recognize multiple epitopes within the target region (aa 277-326) . This multi-epitope recognition can enhance signal amplification for low abundance targets. In contrast, monoclonal antibodies (produced in mouse) offer superior specificity by targeting a single epitope, thus reducing background and cross-reactivity issues . For quantitative applications requiring high reproducibility across experiments, monoclonals demonstrate better lot-to-lot consistency . In Western blot applications with low target expression, polyclonal formats typically provide detection limits in the picogram range, while monoclonals may require nanogram quantities of target protein . When selecting between formats, researchers should prioritize polyclonal antibodies for exploratory studies with potentially low target abundance, and switch to monoclonal formats for standardized, reproducible detection once expression levels are established .

What controls should be included when using Cleaved-CTSA (R326) Antibody in Western blot experiments?

A comprehensive control strategy for Western blot experiments using Cleaved-CTSA (R326) Antibody should include: (1) Positive control: Lysates from cells/tissues known to express cleaved CTSA (activated macrophages or lysosomal fractions often express high levels) ; (2) Negative control: CTSA knockout or knockdown samples; (3) Antibody specificity control: Pre-incubation of the antibody with the immunizing peptide (aa 277-326) should abolish specific binding; (4) Cleavage control: Paired samples with one treated with proteases known to generate the R326 cleavage and one without treatment; (5) Loading control: Probing for house-keeping proteins (β-actin, GAPDH) to ensure equal loading across samples; (6) Molecular weight marker: To confirm the 32 kDa band corresponds to cleaved CTSA; (7) Secondary antibody control: Omitting primary antibody to detect non-specific binding of secondary antibody . Including these controls ensures reliable interpretation of results and helps troubleshoot any unexpected findings.

How can Cleaved-CTSA (R326) Antibody be used to study the relationship between CTSA cleavage and lysosomal function?

To investigate the relationship between CTSA cleavage and lysosomal function using Cleaved-CTSA (R326) Antibody, implement a multi-method experimental approach: First, use subcellular fractionation to isolate lysosomal compartments and analyze cleaved vs. uncleaved CTSA ratios by Western blot . Second, perform immunofluorescence co-localization studies with lysosomal markers (LAMP1, LAMP2) and Cleaved-CTSA (R326) Antibody to map the spatial distribution of cleaved CTSA within lysosomes . Third, manipulate lysosomal pH or protease activity (using inhibitors like E-64, pepstatin A) and monitor changes in CTSA cleavage patterns. Fourth, in cells with galactosialidosis or related lysosomal storage disorders, assess how cleaved CTSA levels correlate with beta-galactosidase and neuraminidase activities, since CTSA forms protective complexes with these enzymes . Finally, use live-cell imaging with pH-sensitive fluorophores alongside immunostaining for cleaved CTSA to track how cleavage events correlate with changes in lysosomal pH and function over time.

What are the key considerations when designing time-course experiments to study CTSA cleavage dynamics?

When designing time-course experiments to study CTSA cleavage dynamics using Cleaved-CTSA (R326) Antibody, several methodological factors require careful consideration: First, establish appropriate time intervals based on the biological process under study (lysosomal maturation typically requires 0-24 hour timepoints, while enzyme complex formation may need shorter intervals) . Second, implement synchronized cellular systems where possible (e.g., pulse-chase experiments with labeled CTSA precursors) to track progression from synthesis to cleavage. Third, simultaneously monitor both uncleaved precursor and cleaved product using antibodies targeting different epitopes, calculating the cleaved/uncleaved ratio at each timepoint for quantitative analysis . Fourth, incorporate metabolic inhibitors at specific timepoints to determine rate-limiting steps in the cleavage process. Fifth, maintain consistent sample processing across all timepoints, particularly controlling proteolysis during extraction to prevent ex vivo cleavage that could confound results. Finally, complement Western blot time-course data with functional assays measuring CTSA carboxypeptidase activity to correlate cleavage status with enzymatic function .

What are common causes of high background when using Cleaved-CTSA (R326) Antibody in immunoassays?

High background when using Cleaved-CTSA (R326) Antibody can result from several factors: (1) Insufficient blocking: Increase blocking time to 2 hours or optimize blocking buffer composition (try 5% BSA instead of milk for phospho-specific applications) ; (2) Antibody concentration: If using polyclonal antibody, high concentrations may increase non-specific binding - try more dilute solutions (1:2000 instead of 1:500) ; (3) Secondary antibody cross-reactivity: Test secondary antibody alone (without primary) to check for direct binding to sample; (4) Sample preparation issues: Incomplete lysis or denaturation may expose hydrophobic regions causing non-specific binding; (5) Buffer composition: High detergent concentrations in wash buffers (>0.1% Tween-20) can reduce specific signal while not adequately removing non-specific binding; (6) Contamination of antibody: Microbial growth in antibody solutions can cause background - check for cloudiness and filter if necessary; (7) Over-development: Particularly in chemiluminescent detection systems, extended exposure times amplify background signals . Systematic troubleshooting by adjusting each parameter individually will help identify and resolve the specific cause.

How can I optimize detection of cleaved CTSA in formalin-fixed paraffin-embedded (FFPE) tissue samples?

Optimizing detection of cleaved CTSA in FFPE samples requires specialized techniques due to the denatured state of proteins. First, ensure complete antigen retrieval using citrate buffer (pH 6.0) with heat treatment (95-100°C for 20-30 minutes) . The Cleaved-CTSA (R326) Antibody targets a linear epitope (aa 277-326), which may be advantageous in FFPE detection compared to antibodies targeting conformational epitopes . Use a detection system with signal amplification (e.g., tyramide signal amplification) to enhance sensitivity for low-abundance targets. Optimize antibody concentration through titration experiments, typically starting with higher concentrations (1:100-1:200) than used for Western blot . For multi-color immunohistochemistry, use antibodies raised in different species or directly conjugated primary antibodies to avoid cross-reactivity. Additionally, incorporate appropriate positive controls (tissues known to express cleaved CTSA) and negative controls (peptide competition) in each experiment . For decalcified specimens, extended antibody incubation times (overnight at 4°C) may improve penetration and detection sensitivity.

What strategies can be employed to reduce the impact of freeze-thaw cycles on Cleaved-CTSA (R326) Antibody performance?

To minimize performance degradation from freeze-thaw cycles, implement the following protocol: Upon receipt, aliquot the antibody into single-use volumes (10-20 μL) in sterile microcentrifuge tubes . For dilute antibody solutions, consider adding carrier protein (BSA, 0.1-1%) to prevent adsorption to tube walls. Use screw-cap tubes with O-rings to prevent evaporation during long-term storage. Label each aliquot with dilution factor, date, and number of previous freeze-thaw cycles. Store aliquots at -20°C for regular use or -80°C for extended storage . When thawing, place on ice and allow to thaw gradually rather than using rapid warming methods. Once thawed, mix gently by pipetting or flicking (avoid vortexing which can denature antibodies). After use, immediately return unused portion to -20°C. For working dilutions, prepare fresh on the day of experiment rather than storing diluted antibody. If an aliquot has undergone more than 5 freeze-thaw cycles, perform a validation experiment comparing to a fresh aliquot before using in critical experiments .

How can Cleaved-CTSA (R326) Antibody be used to study lysosomal storage disorders like galactosialidosis?

Cleaved-CTSA (R326) Antibody offers valuable applications for studying galactosialidosis and related lysosomal storage disorders. First, use the antibody to quantify the ratio of cleaved (active) to uncleaved (precursor) CTSA in patient-derived fibroblasts, comparing with healthy controls to establish correlation between cleavage efficiency and disease severity . Second, perform co-immunoprecipitation experiments with Cleaved-CTSA (R326) Antibody to isolate and characterize the multienzyme complex formation between cleaved CTSA, beta-galactosidase, and neuraminidase, which is disrupted in galactosialidosis . Third, use the antibody in pulse-chase experiments to track CTSA processing kinetics in normal vs. disease cells, revealing potential processing defects . Fourth, in animal models of galactosialidosis, use immunohistochemistry with this antibody to map tissue-specific accumulation patterns of uncleaved vs. cleaved CTSA. Finally, in therapeutic development studies (enzyme replacement, gene therapy), the antibody can monitor restoration of normal CTSA processing as a biomarker of treatment efficacy.

What are effective protocols for using Cleaved-CTSA (R326) Antibody in immune cell function studies?

For studying immune cell functions using Cleaved-CTSA (R326) Antibody, implement the following protocol adaptations: First, for flow cytometry applications, fix cells with 4% paraformaldehyde followed by permeabilization with 0.1% saponin to access intracellular CTSA . Use a 1:200 dilution of primary antibody and appropriate fluorophore-conjugated secondary antibody . Second, for studying CTSA cleavage during immune cell activation, isolate peripheral blood mononuclear cells (PBMCs) and stimulate with appropriate activators (LPS, PMA/ionomycin, etc.) for various time periods (0-48h) . Process samples for Western blot using the antibody to track cleavage kinetics. Third, combine with functional assays (cytokine ELISAs, migration assays) to correlate CTSA cleavage status with specific immune functions . Fourth, for tissue macrophage analysis, optimize antigen retrieval conditions (citrate buffer pH 6.0, 95°C for 20 minutes) for FFPE sections before antibody application . Finally, in co-culture systems (e.g., macrophages with cancer cells), use fluorescent labeling of cell populations combined with Cleaved-CTSA (R326) Antibody staining to track cell-specific changes in CTSA processing during cellular interactions .

What experimental approaches can integrate Cleaved-CTSA (R326) Antibody in cancer research studies?

Integrating Cleaved-CTSA (R326) Antibody into cancer research requires specialized experimental designs: First, perform comparative analysis of cleaved CTSA levels in tumor tissues versus matched normal tissues using immunohistochemistry or Western blot, correlating with clinical parameters and patient outcomes . Second, investigate tumor microenvironment interactions by dual staining for cleaved CTSA and cell-type markers (CD68 for macrophages, CD31 for endothelial cells) to identify which cells within tumors express activated CTSA . Third, in metastasis studies, analyze whether CTSA cleavage patterns differ between primary tumors and metastatic sites, potentially revealing proteolytic signatures associated with invasive phenotypes. Fourth, for drug development, use the antibody to monitor how treatments affect CTSA processing in cancer cells, particularly for therapies targeting lysosomal function or autophagy . Fifth, in combination with proteomics approaches, use immunoprecipitation with this antibody to identify novel substrate proteins cleaved by activated CTSA in tumor contexts. Finally, develop prognostic assays based on quantitative measurement of cleaved CTSA in tumor biopsies, potentially identifying patient subgroups with distinct clinical trajectories.

What are the detailed specifications for different formats of Cleaved-CTSA (R326) Antibody available for research?

The detailed specifications for Cleaved-CTSA (R326) Antibody formats are outlined below:

What is the molecular basis for the R326 cleavage site in CTSA and how does the antibody specifically recognize this region?

The R326 cleavage site in CTSA represents a critical processing point in the maturation of this lysosomal enzyme. In the molecular pathway, CTSA is synthesized as a 54 kDa precursor that undergoes proteolytic processing in the lysosome to generate the mature active form consisting of two chains: a 32 kDa heavy chain and a 20 kDa light chain . The specific cleavage occurs adjacent to arginine 326 (R326 in humans, R314 in mice) , which is located in a protease-accessible region of the protein. The Cleaved-CTSA (R326) Antibody was specifically generated against a synthetic peptide corresponding to amino acids 277-326 of human PPGB . This antibody specifically recognizes the neo-epitope created when the protein is cleaved, as the peptide used for immunization contains the C-terminal sequence of the heavy chain that becomes exposed only after cleavage . This specificity is confirmed by the antibody's ability to detect "endogenous levels of fragment of activated Cathepsin A 32k protein resulting from cleavage adjacent to R326" . The antibody likely recognizes a conformational change or newly exposed linear epitope that is inaccessible in the intact precursor form, allowing researchers to specifically monitor the activation status of CTSA in biological samples.

What are the recommended dilution ranges for different applications of Cleaved-CTSA (R326) Antibody based on empirical testing?

Based on empirical testing, the recommended dilution ranges for different applications of Cleaved-CTSA (R326) Antibody are:

*Note: Applications marked with asterisk have limited validation data and may require extensive optimization by the end-user .

How does Cleaved-CTSA (R326) Antibody compare with other antibodies targeting different regions of CTSA?

Comparing Cleaved-CTSA (R326) Antibody with other CTSA-targeting antibodies reveals distinct advantages and limitations:

This comparison demonstrates that Cleaved-CTSA (R326) Antibody fills a specific niche in CTSA research by providing information about the activation state rather than just presence/absence of the protein, making it uniquely valuable for studies focused on CTSA processing and function .

What are the relative advantages and disadvantages of using Cleaved-CTSA (R326) Antibody compared to activity-based assays for measuring CTSA function?

The relative advantages and disadvantages of Cleaved-CTSA (R326) Antibody versus activity-based assays for measuring CTSA function represent important considerations for experimental design:

For comprehensive CTSA characterization, combining both approaches is optimal: use Cleaved-CTSA (R326) Antibody to determine cleavage/activation status , then confirm functional consequences through activity-based assays, creating a more complete picture of CTSA biology in experimental systems.

How do assay results with Cleaved-CTSA (R326) Antibody correlate with functional measures of lysosomal activity?

The correlation between Cleaved-CTSA (R326) Antibody assay results and functional lysosomal activity measures presents a complex relationship requiring careful interpretation:

These correlations highlight that while Cleaved-CTSA (R326) Antibody provides valuable information about CTSA processing status , comprehensive assessment of lysosomal function requires multiple complementary approaches. The antibody serves as an important tool within a broader experimental toolkit for understanding lysosomal biology.