StPat14K07.03 Antibody

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Product Specs

Buffer
Preservative: 0.03% Proclin 300
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Form
Liquid
Lead Time
Made-to-order (14-16 weeks)
Synonyms
StPat14K07.03 antibody; Patatin-02 antibody; EC 3.1.1.- antibody; Patatin group A-1 antibody
Target Names
StPat14K07.03
Uniprot No.

Target Background

Function
Probable lipolytic acyl hydrolase (LAH), an activity believed to be involved in the response of tubers to pathogens.
Database Links

UniGene: Stu.20031

Protein Families
Patatin family
Subcellular Location
Vacuole.
Tissue Specificity
Tuber and stolon.

Q&A

What is the specificity profile of ST14/Matriptase antibodies in various experimental systems?

ST14/Matriptase antibodies demonstrate high specificity across multiple experimental platforms when properly validated. For flow cytometry applications, anti-human Matriptase/ST14 catalytic domain antibodies successfully detect the protein in prostate cancer cell lines such as PC-3, with clear differentiation from control antibody staining patterns . This specificity extends to Western blot applications where matriptase can be distinguished from other serine proteases. Detection typically requires proper cell fixation with paraformaldehyde and permeabilization with agents like saponin to facilitate intracellular staining . Researchers should validate antibody specificity in their specific experimental system by including appropriate controls such as isotype antibodies or examining samples from knockout models (e.g., St14–/– tissues), which can confirm the absence of signal and thus authenticate antibody specificity .

How do matriptase/ST14 antibodies perform in detecting different matriptase forms?

Matriptase/ST14 antibodies can effectively distinguish between the zymogen (inactive precursor) and activated forms of the protease in various tissue samples. In capillary electrophoresis experiments, these antibodies have successfully identified both forms across multiple tissues including skin, kidney, lung, and intestine . The zymogen appears at higher molecular weight positions compared to the activated forms, which can be indicated using separate markers (filled arrows for zymogens, open arrows for activated forms) . This differential detection capability is particularly valuable when examining protease activation cascades, as demonstrated in studies examining the relationship between matriptase and its substrate prostasin. For optimal detection of both forms, researchers should carefully select antibodies specifically validated for distinguishing between zymogen and activated states rather than general anti-matriptase antibodies.

What detection methods are compatible with ST14/matriptase antibodies?

ST14/matriptase antibodies have been successfully employed across multiple detection platforms with varying sensitivities. These include:

  • Flow cytometry - Allowing quantitative analysis of matriptase expression at the cellular level with successful detection demonstrated in prostate cancer cell lines using fluorophore-conjugated secondary antibodies (e.g., NorthernLights™ 557-conjugated Anti-Sheep IgG)

  • Capillary electrophoresis/Simple Western - Enabling high-resolution separation and quantification of matriptase forms in tissue extracts with detection sensitive enough to distinguish between zymogen and activated forms

  • Immunohistochemistry - Although not explicitly detailed in the provided data, antibodies against proteases are commonly used for tissue localization studies

Each method requires specific optimization, including appropriate sample preparation (fixation/permeabilization for flow cytometry), antibody concentration determination through titration experiments, and selection of compatible detection systems (fluorophores, enzyme conjugates) based on experimental design requirements.

How should researchers optimize detection of matriptase/ST14 in complex tissue samples?

Optimizing matriptase/ST14 detection in complex tissue samples requires careful attention to several experimental parameters. Based on published protocols, researchers should:

  • Implement appropriate protein extraction methods tailored to the specific tissue type. For instance, when working with skin, kidney, lung, or intestinal tissues, protein extraction requires careful homogenization to prevent protease degradation while maintaining native protein structures .

  • Consider sample preparation variations between tissues - the protein extraction protocol that works optimally for skin may require modification for intestinal samples due to different protease environments and matrix compositions.

  • Include tissue-appropriate controls in experimental design. The ideal approach incorporates tissues from wild-type (St14+/+), heterozygous, and knockout (St14–/–) animals to establish detection baselines .

  • Normalize protein loading using housekeeping proteins such as β-actin to ensure quantitative comparisons between samples are valid .

  • For detecting both zymogen and activated forms of matriptase, separate samples under non-reducing conditions to preserve disulfide bonds that may distinguish different conformational states of the protein.

The detection sensitivity can be further enhanced by employing signal amplification systems appropriate to the chosen method. For example, in Western blot applications, enhanced chemiluminescence systems with extended exposure times may improve detection of low-abundance activated forms.

What are the key considerations when studying matriptase-prostasin interaction using antibody-based approaches?

The matriptase-prostasin proteolytic cascade represents a critical pathway in epithelial biology, requiring specialized experimental approaches for accurate characterization:

  • Antibody selection is crucial - researchers should use antibodies capable of distinguishing between zymogen and activated forms of both matriptase and prostasin to properly characterize the activation cascade . This typically requires antibodies targeting specific epitopes exposed or hidden in different activation states.

  • Experimental design should incorporate appropriate genetic models to validate interactions. Studies have successfully utilized St14zym/zym (zymogen-locked matriptase), St14+/+, and St14–/– animal models alongside Prss8–/– (prostasin null) and Prss8zym/zym (zymogen-locked prostasin) models to comprehensively map this proteolytic relationship .

  • Quantitative analysis approaches should measure both absolute levels and relative ratios of different forms. For example, calculating the ratio of activated prostasin to total prostasin provides mechanistic insight into the functional consequences of matriptase variants .

  • Statistical analysis must be appropriate for the data structure - one-way ANOVA with appropriate post-hoc tests (as demonstrated in published studies with P = 0.0011) can determine significance in activation differences between genetic variants .

This experimental framework allows researchers to establish causal relationships between matriptase activity and downstream substrate processing, particularly important when investigating epithelial barrier formation and function.

How can researchers validate antibody performance in matriptase/ST14 detection across different species?

Cross-species validation of matriptase/ST14 antibodies requires systematic evaluation across multiple parameters:

  • Sequence homology analysis - Before experimental validation, researchers should analyze sequence conservation in the antibody epitope region between species. Matriptase/ST14 shows conserved domains across mammals, but epitope-specific variations may affect antibody binding.

  • Sequential validation across species - Begin with positive control samples from the species for which the antibody was developed (e.g., human-specific antibodies with human cell lines), then test progressively in related species.

  • Implementation of knockout controls - Whenever possible, include tissue samples from ST14 knockout animals of the target species to confirm signal specificity .

  • Cross-reactivity testing - Test for potential cross-reactivity with closely related serine proteases such as hepsin or TMPRSS2 that share structural similarities with matriptase.

  • Parallel technique validation - Confirm antibody performance across multiple techniques (Western blot, immunohistochemistry, flow cytometry) as species cross-reactivity may vary between applications due to differences in epitope accessibility.

When reporting cross-species reactivity, researchers should explicitly document all validation steps performed and provide quantitative metrics of binding efficiency compared to the original target species.

How can researchers track matriptase/ST14 activation dynamics in live cell systems?

Investigating matriptase/ST14 activation dynamics in live cell systems presents significant technical challenges but can be approached through several advanced methodologies:

  • Fluorescent reporter systems - Engineer cells to express matriptase fused with split fluorescent proteins or FRET (Förster Resonance Energy Transfer) pairs that change fluorescence properties upon protease activation. This allows real-time monitoring of activation events.

  • Activity-based probes - Develop or utilize available activity-based probes that selectively bind to the active site of activated matriptase but not the zymogen form. These probes can be conjugated to fluorophores for live imaging applications.

  • Antibody-based approaches - While conventional antibodies require cell fixation, specially developed non-perturbing antibody fragments (Fabs) directed against conformation-specific epitopes of matriptase can be adapted for live cell imaging when conjugated to cell-permeable fluorophores.

  • Temporal sampling with rapid fixation - When live imaging is not feasible, researchers can implement time-course experiments with rapid fixation protocols to "freeze" activation states at defined timepoints, followed by immunostaining with antibodies that distinguish between zymogen and activated forms .

Each approach requires careful validation, including confirmation that the detection method itself does not alter the activation dynamics being studied. Controls should include protease inhibitor treatments and catalytically inactive matriptase mutants to establish baseline signals.

What strategies can resolve contradictory findings when using different matriptase/ST14 antibodies?

Contradictory findings when using different matriptase/ST14 antibodies are not uncommon and require systematic troubleshooting:

  • Epitope mapping analysis - Different antibodies may recognize distinct epitopes on matriptase that are differentially exposed under various experimental conditions or in different matriptase conformational states. Researchers should document the specific epitope regions targeted by each antibody.

  • Validation using genetic models - Results from different antibodies should be compared against samples from matriptase knockout (St14–/–) and zymogen-locked (St14zym/zym) models to determine which antibody most accurately reflects the true biological state .

  • Orthogonal method verification - Complement antibody-based detection with non-antibody methods such as mass spectrometry-based proteomics or activity assays to resolve discrepancies.

  • Standardized sample preparation - Variations in fixation, permeabilization, or protein extraction protocols can significantly impact epitope availability. Researchers should implement identical sample preparation protocols when comparing antibodies.

  • Antibody validation consortium approach - When facing persistent contradictions, consider establishing a multi-laboratory validation effort where different antibodies are tested under standardized conditions across different research groups.

Documenting and publishing these comparative analyses contributes valuable information to the field regarding antibody reliability and specificity boundaries.

How can protease-antibody complexes impact functional measurements of matriptase/ST14 activity?

The formation of protease-antibody complexes can significantly influence functional measurements of matriptase/ST14, creating important experimental considerations:

  • Allosteric effects - Antibody binding, even to sites distant from the catalytic domain, can induce conformational changes that alter enzyme kinetics. Researchers should compare activity measurements before and after antibody addition to detect such effects.

  • Active site occlusion - Antibodies targeting epitopes near or within the catalytic domain may directly interfere with substrate access, producing apparent inhibition that does not reflect physiological regulation .

  • Stabilization effects - Some antibodies may preferentially stabilize either the zymogen or active conformation of matriptase, artificially shifting the equilibrium between these states in experimental systems.

  • Complex formation impacts - In proteolytic cascades like the matriptase-prostasin system, antibody binding to matriptase may alter its ability to interact with and activate downstream substrates, complicating interpretation of cascade dynamics .

To mitigate these issues, researchers should:

  • Use Fab fragments rather than intact antibodies when measuring enzyme kinetics

  • Implement control experiments with non-specific antibodies of the same isotype

  • Validate findings with multiple antibodies targeting different epitopes

  • Consider developing activity-based assays that don't require antibody binding during the activity measurement phase

How should researchers quantify and interpret matriptase/ST14 activation states in different physiological contexts?

Accurate quantification and interpretation of matriptase/ST14 activation states requires careful methodological consideration:

  • Standardized quantification methods - Researchers should employ densitometric analysis of separated protein bands from capillary electrophoresis or traditional Western blots, clearly distinguishing between zymogen (filled arrows) and activated forms (open arrows) as demonstrated in published protocols .

  • Activation ratio calculations - Rather than absolute values alone, calculate the ratio of activated matriptase to total matriptase (activated plus zymogen forms) to normalize for expression level variations between samples .

  • Physiological context calibration - Different tissues exhibit distinct baseline activation states. For example, skin samples typically show different matriptase activation patterns compared to intestinal samples, requiring tissue-specific interpretation frameworks .

  • Statistical analysis approaches - When comparing activation states across experimental groups (e.g., wild-type vs. zymogen-locked models), apply appropriate statistical tests such as one-way ANOVA with post-hoc analysis to determine significance, reporting both p-values and standard deviations .

  • Technical replicate integration - Combine data from multiple technical replicates (n ≥ 3) and biological replicates to establish confidence intervals for activation measurements.

This quantitative framework allows meaningful comparisons across different physiological and pathological states, enabling insights into how matriptase activation contributes to biological processes.

What are the best practices for interpreting colocalization of matriptase/ST14 with potential substrates?

Interpreting colocalization of matriptase/ST14 with potential substrates requires rigorous analytical approaches beyond simple overlay observations:

  • Quantitative colocalization metrics - Move beyond visual assessment to calculate statistical measures such as Pearson's correlation coefficient, Manders' overlap coefficient, or intensity correlation quotient to quantify the degree of spatial overlap between matriptase and putative substrates.

  • Resolution considerations - Acknowledge the resolution limits of the imaging modality used. Conventional light microscopy (resolution ~200-300 nm) may suggest colocalization when proteins are merely in proximity rather than directly interacting.

  • Functional validation requirements - Complement colocalization data with functional evidence of enzyme-substrate relationships. This can be achieved by monitoring substrate processing in the presence of wild-type versus catalytically inactive matriptase, or in systems with matriptase knockdown/knockout .

  • Temporal dynamics analysis - Static colocalization snapshots may miss transient interactions. Where possible, implement time-lapse imaging to capture the dynamic nature of enzyme-substrate interactions.

  • Proximity ligation assays - For more definitive evidence of close proximity (≤40 nm), implement proximity ligation assays that generate fluorescent signals only when two proteins are within molecular interaction distance.

When interpreting such data, researchers should clearly distinguish between evidence of spatial proximity and confirmed functional interactions, acknowledging that colocalization is necessary but not sufficient to establish enzyme-substrate relationships.

How can researchers differentiate between direct and indirect effects of matriptase/ST14 in proteolytic cascades?

Differentiating between direct and indirect effects of matriptase/ST14 in proteolytic cascades presents significant analytical challenges:

  • Recombinant protein interaction studies - Use purified recombinant matriptase and putative substrate proteins in cell-free systems to test direct proteolytic activity, establishing kinetic parameters such as Km and kcat values.

  • Zymogen-locked mutant comparisons - Employ genetic models expressing catalytically inactive matriptase (St14zym/zym) to distinguish between scaffolding functions and proteolytic activities of matriptase . This approach has been successfully used to demonstrate matriptase's role in prostasin activation, where zymogen-locked matriptase still permits some prostasin processing but at significantly reduced efficiency compared to wild-type .

  • Temporal sequence resolution - Implement tightly controlled time-course experiments to establish the sequence of activation events, helping to distinguish primary (direct) from secondary (indirect) proteolytic events.

  • Targeted inhibition approaches - Use highly selective protease inhibitors or blocking antibodies against specific components of the cascade to isolate their individual contributions.

  • Quantitative pathway modeling - Develop mathematical models of the proteolytic network incorporating experimentally determined rate constants to predict and test how perturbation of matriptase activity propagates through the system.

The combined evidence from these approaches allows researchers to construct mechanistic models that accurately represent the direct and indirect roles of matriptase in proteolytic networks, as exemplified by the matriptase-prostasin system studied in epithelial biology .

What emerging technologies might enhance antibody-based detection of matriptase/ST14?

Several emerging technologies show promise for advancing antibody-based detection of matriptase/ST14 and related proteases:

  • Single-molecule detection platforms - Technologies such as single-molecule FRET or super-resolution microscopy can reveal matriptase activation events at unprecedented spatial resolution, potentially capturing individual protease molecules transitioning between zymogen and active states.

  • Quantum dot-labeled lateral flow immunoassays (QD-LFIA) - Similar to approaches used in other antibody detection systems, QD-labeled antibodies could provide enhanced sensitivity and quantitative detection capabilities for matriptase in complex biological samples .

  • Mass cytometry (CyTOF) - This technology allows simultaneous detection of dozens of protein markers at the single-cell level, potentially enabling comprehensive mapping of matriptase in relation to multiple components of proteolytic cascades.

  • Multiplex biosensor arrays - Development of antibody-based biosensor arrays could enable real-time monitoring of multiple proteases simultaneously, capturing the dynamics of entire proteolytic networks rather than individual components.

  • Antibody engineering - Creation of conformation-specific recombinant antibodies or antibody fragments with enhanced specificity for different matriptase states through directed evolution approaches.

These technological advances may help resolve current limitations in detecting low-abundance activated forms of matriptase and provide more dynamic information about proteolytic processes in complex biological systems.

How might long-term stability data for other antibodies inform storage and handling of matriptase/ST14 antibodies?

Insights from long-term antibody stability studies can inform optimal storage and handling protocols for matriptase/ST14 antibodies:

  • Temperature storage considerations - Based on studies of antibody longevity in serum samples, maintaining storage at -80°C with minimal freeze-thaw cycles likely preserves optimal binding characteristics . Studies tracking antibodies for over 400 days demonstrate that proper storage prevents significant degradation of antibody function .

  • Buffer composition impact - Storage buffers containing stabilizing agents such as glycerol (typically 30-50%) and carrier proteins may enhance long-term stability by preventing aggregation and surface adsorption.

  • Aliquoting strategies - Preparing multiple small-volume aliquots during initial antibody processing minimizes exposure to repeated freeze-thaw cycles, which studies have shown can significantly impact antibody performance over time .

  • Stability testing protocols - Implementing periodic quality control testing using standard antigens can track potential degradation over time. Similar to approaches used in tracking SARS-CoV-2 antibodies, researchers can establish baseline titers and monitor changes over storage duration .

  • Lyophilization considerations - For extremely long-term storage, lyophilization (freeze-drying) may offer advantages over liquid storage, though reconstitution protocols must be carefully optimized to maintain antibody function.

By implementing these evidence-based storage and handling practices, researchers can maximize the functional lifespan of valuable matriptase/ST14 antibody reagents and ensure experimental reproducibility over extended research programs.

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