PRSS35 Antibody

What is PRSS35 and what biological functions does it serve?

PRSS35, or serine protease 35, functions as a key player in proteolytic pathways that regulate cellular function and disease progression. It has emerged as a promising research target for studies involving cancer, inflammation, and neurodegenerative disorders. Recent evidence specifically identifies PRSS35 as a tumor suppressor in hepatocellular carcinoma (HCC), where it suppresses CXCL2-mediated neutrophil extracellular traps . As a secreted protease, PRSS35 undergoes processing via cleavage by proprotein convertases to become active, after which it targets proteins containing tandem lysine (KK) recognition motifs . This activity positions PRSS35 as an important regulatory element in the extracellular microenvironment.

Which applications are PRSS35 antibodies most commonly used for?

PRSS35 antibodies are primarily employed in Western blotting, ELISA, and immunohistochemistry applications . Western blotting represents the most validated application, with recommended dilutions typically ranging from 1:1000 to 1:5000 . These antibodies enable accurate detection and quantification of PRSS35 in various cellular contexts, making them valuable for investigating molecular biology, cell signaling pathways, and cancer research . For immunohistochemistry, dilutions between 1:20 and 1:200 are generally recommended to achieve optimal staining with minimal background .

What sample types work best with PRSS35 antibodies?

PRSS35 antibodies have demonstrated successful detection in multiple sample types. Mouse and rat testis tissues serve as positive control samples for validating antibody performance . In human samples, PRSS35 has been successfully detected in liver tissues and serum samples . When working with clinical specimens, researchers should note that PRSS35 shows differential expression between healthy and diseased states - particularly in HCC, where both tissue and serum levels are significantly reduced compared to normal controls . This differential expression pattern makes PRSS35 a potential biomarker candidate for certain pathological conditions.

How should I optimize Western blot protocols for PRSS35 detection?

For optimal Western blot detection of PRSS35, consider the following methodological approach:

• Sample preparation: Given PRSS35's presence in both cellular and secreted forms, analyze both cell lysates and conditioned media

• Protein separation: Use gradient gels (4-15%) to effectively resolve the full-length PRSS35 (~45 kDa) and its shorter variants

• Transfer conditions: Employ wet transfer methods with methanol-containing buffer for efficient transfer of all PRSS35 forms

• Antibody selection: Choose antibodies based on your target region of interest, as different antibodies detect distinct forms:

  • N-terminal antibodies detect full-length PRSS35

  • C-terminal antibodies capture processed forms

• Blocking and incubation: Use 5% non-fat milk in TBST for blocking, with primary antibody dilutions at 1:1000-1:5000

• Detection: For enhanced sensitivity, particularly with secreted forms, consider using chemiluminescent substrates with extended exposure times

When interpreting results, note that PRSS35 appears in multiple molecular weight forms due to post-translational processing, with the secreted form (SF-PRSS35) having significantly lower molecular weight than full-length (FL-PRSS35) .

What are the critical considerations for PRSS35 immunohistochemistry experiments?

When performing immunohistochemistry for PRSS35, researchers should implement the following methodological approach:

• Fixation: Use 10% neutral buffered formalin for tissue fixation to preserve antigen integrity

• Antigen retrieval: Employ heat-induced epitope retrieval in citrate buffer (pH 6.0) to expose PRSS35 epitopes

• Antibody selection: Choose antibodies targeting specific regions based on research objectives:

  • For total PRSS35 detection, use C-terminal antibodies

  • For distinguishing processed forms, employ region-specific antibodies

• Antibody dilution: Start with 1:20-1:200 dilutions and optimize based on signal-to-noise ratio

• Detection system: Utilize polymer-based detection systems for enhanced sensitivity

• Controls: Include both positive (testis tissue) and negative controls in each experiment

• Counterstaining: Use light hematoxylin counterstaining to maintain visibility of PRSS35 signal

For quantitative analysis, consider using digital pathology tools to measure staining intensity across tissue sections, particularly when comparing expression levels between normal and pathological samples, as PRSS35 shows gradual reduction with increasing stages of HCC development .

How can I differentiate between full-length and processed forms of PRSS35?

Distinguishing between full-length and processed PRSS35 forms requires a strategic approach:

• Antibody panel selection: Utilize multiple antibodies targeting different regions:

  • N-terminal (N-PRSS35)

  • Middle region (M-PRSS35)

  • C-terminal (C-PRSS35)

• Molecular weight analysis: Compare observed bands with expected molecular weights:

  PRSS35 Form	Approximate MW	Detected By
  Full-length (FL-PRSS35)	~45 kDa	N, M, C antibodies
  Secreted form (SF-PRSS35)	Significantly lower	Primarily C antibodies
  Processed variants	Multiple bands	Varies by fragment

• Subcellular fractionation: Separate cellular compartments to determine localization:

  • Cell lysates contain primarily FL-PRSS35

  • Culture medium/extracellular fractions contain SF-PRSS35

• Mass spectrometry validation: Confirm band identity through peptide sequencing, which has verified that shorter bands detected by SDS-PAGE contain PRSS35 peptides

• Western blot interpretation: When analyzing samples, note that FL-PRSS35 is predominantly found in cell lysates, while SF-PRSS35 accumulates in culture medium of PRSS35-expressing cells .

What approaches should I use to study PRSS35's tumor suppressor function in cancer models?

To investigate PRSS35's tumor suppressor functions in cancer models, implement these methodological strategies:

• Expression analysis in clinical samples:

  • Compare PRSS35 levels in matched tumor/normal tissues using Western blotting

  • Develop ELISA-based quantification of serum PRSS35 levels as potential biomarkers

  • Correlate expression with patient survival data and clinical parameters

• In vitro functional studies:

  • Establish PRSS35 overexpression and knockdown systems in relevant cell lines

  • Assess effects on cell proliferation, migration, invasion, and colony formation

  • Investigate PRSS35-mediated cleavage of potential substrates like CXCL2

• In vivo tumor models:

  • Xenograft models: Inject PRSS35-overexpressing cells (e.g., HepG2) into nude mice

  • Spontaneous HCC model: Use hydrodynamic injection of YAP-5SA with PRSS35 modulation

  • PRSS35 knockout mice: Compare tumor development in WT vs. KO animals

• Mechanism investigation:

  • Analyze neutrophil recruitment and extracellular trap formation

  • Quantify CXCL2 levels and processing

  • Study the HNF4A-PRSS35 regulatory axis using luciferase reporter assays

• Therapeutic potential assessment:

  • Test recombinant PRSS35 administration effects on established tumors

  • Develop strategies to enhance endogenous PRSS35 expression

This comprehensive approach has revealed that PRSS35 overexpression significantly suppresses HCC growth in multiple mouse models, while PRSS35 knockout accelerates liver cancer development .

How can I develop and validate a custom ELISA for PRSS35 quantification in clinical samples?

Developing a robust ELISA for PRSS35 quantification in clinical samples involves these methodological steps:

• Antibody pair selection:

  • Choose capture and detection antibodies targeting different epitopes

  • Consider using dual N-terminal antibodies for specific detection

  • Validate antibody specificity using recombinant PRSS35 protein

• ELISA protocol optimization:

  Parameter	Recommended Approach
  Plate coating	1-10 μg/ml capture antibody in carbonate buffer (pH 9.6)
  Blocking	1-5% BSA or non-fat milk in PBS-T
  Sample dilution	Optimize based on expected concentration range
  Standard curve	Use recombinant PRSS35 protein (5-500 ng/ml)
  Detection	HRP or biotin-conjugated detection antibody
  Substrate	TMB with appropriate development time

• Validation parameters:

  • Analytical sensitivity: Determine limit of detection (LOD) and quantification (LOQ)

  • Specificity: Test for cross-reactivity with related proteases

  • Precision: Assess intra- and inter-assay coefficient of variation (CV)

  • Recovery: Spike known amounts of PRSS35 into samples

  • Parallelism: Verify similar detection across dilutions

• Clinical sample analysis:

  • Establish reference ranges in healthy controls

  • Compare levels in disease states (e.g., HCC patients)

  • Correlate with clinical parameters and outcomes

This approach has been successfully implemented to demonstrate significantly lower serum PRSS35 levels in HCC patients compared to normal subjects, suggesting its potential as a prognostic biomarker .

Why might I observe multiple bands when performing Western blot for PRSS35?

The observation of multiple bands during PRSS35 Western blotting is an expected phenomenon with specific biological significance. Multiple bands result from:

• Post-translational processing: PRSS35 undergoes cleavage by proprotein convertases, generating various shorter forms

• Antibody specificity: Different antibodies (N-terminal, middle-region, or C-terminal) detect distinct fragments

• Sample preparation: Different extraction methods may preserve or disrupt various processed forms

To properly interpret these patterns:

• Full-length PRSS35 appears at approximately 45 kDa in cell lysates

• Secreted PRSS35 forms have significantly lower molecular weights

• Multiple short forms enrich in culture medium of PRSS35-expressing cells

This is not a technical artifact but reflects the biological reality of PRSS35 processing. Mass spectrometry analysis has confirmed that these shorter bands indeed contain PRSS35 peptides . When troubleshooting, compare your observed pattern with expected processing forms and consider using antibodies targeting different regions to gain a complete picture of PRSS35 expression and processing.

What controls should I include when studying PRSS35 in different experimental models?

When studying PRSS35 across experimental models, implement a comprehensive control strategy:

• Positive tissue controls:

  • Mouse and rat testis tissues have confirmed PRSS35 expression

  • Normal human liver tissue (for HCC-related studies)

• Cell line controls:

  • THLE3 cells (high PRSS35 expression)

  • HCC cell lines like PLC, HepG2, and Hep3B (low PRSS35 expression)

• Expression controls:

  • PRSS35 overexpression system (positive control)

  • PRSS35 knockout or knockdown system (negative control)

  • Empty vector transfection (baseline control)

• Antibody controls:

  • Primary antibody omission

  • Isotype control antibody (matching IgG)

  • Pre-absorption with immunizing peptide

• Processing controls:

  • Recombinant PRSS35 protein standards

  • Protease inhibitor treatment to block processing

• Cross-species consideration:

  • Human PRSS35 for clinical studies

  • Mouse PRSS35 for murine models, noting functional conservation

This comprehensive control strategy ensures reliable data interpretation across different experimental systems and helps distinguish biological variations from technical artifacts.

How can I combine PRSS35 antibody techniques with proteomics to identify novel substrates?

Integrating PRSS35 antibody techniques with proteomics to identify novel substrates involves this methodological workflow:

• Substrate prediction:

  • Analyze potential substrates for PRSS35 recognition motifs (e.g., tandem lysine KK motifs)

  • Perform in silico screening of secreted proteins containing these motifs

• Differential secretome analysis:

  • Implement SILAC (Stable Isotope Labeling with Amino acids in Cell culture) proteomics

  • Compare secretomes from control vs. PRSS35-overexpressing cells

  • Focus on proteins decreased in abundance but not in transcription level

• Immunoprecipitation-mass spectrometry:

  • Use PRSS35 antibodies to pull down PRSS35 and interacting proteins

  • Perform mass spectrometry to identify co-precipitated proteins

  • Validate interactions through reverse immunoprecipitation

• In vitro cleavage assays:

  • Express and purify recombinant candidate substrates

  • Incubate with active PRSS35

  • Analyze cleavage products by SDS-PAGE and mass spectrometry

• Functional validation:

  • Mutate potential cleavage sites in candidate substrates

  • Assess resistance to PRSS35-mediated degradation

  • Evaluate functional consequences of blocked processing

This integrated approach has successfully identified CXCL2 as a PRSS35 substrate, demonstrating how PRSS35 cleaves CXCL2 at its tandem lysine recognition motif, ultimately suppressing neutrophil recruitment and tumor progression .

What approaches should I use to study PRSS35 regulation and expression in different tissues?

To comprehensively investigate PRSS35 regulation and expression across tissues, implement this multi-modal approach:

• Transcriptional regulation analysis:

  • Identify potential transcription factor binding sites in PRSS35 promoter using bioinformatics tools

  • Validate regulatory elements through luciferase reporter assays

  • Confirm transcription factor binding using ChIP (Chromatin Immunoprecipitation)

• Tissue expression profiling:

  • Perform immunohistochemistry on tissue microarrays using optimized protocols (1:20-1:200 dilution)

  • Quantify differential expression using digital pathology tools

  • Correlate expression patterns with pathological features

• Single-cell analysis:

  • Implement single-cell RNA sequencing to identify cell types expressing PRSS35

  • Validate with immunofluorescence co-staining using cell-type markers

  • Map expression changes during disease progression

• Epigenetic regulation:

  • Analyze DNA methylation patterns at PRSS35 promoter

  • Assess histone modifications using ChIP-seq

  • Evaluate effects of epigenetic modifiers on PRSS35 expression

• Post-transcriptional regulation:

  • Identify miRNAs targeting PRSS35 mRNA

  • Validate through luciferase reporter assays with miRNA binding site mutations

  • Assess mRNA stability and translation efficiency

The regulation of PRSS35 by HNF4A represents an example of this approach, where HNF4A response elements in the PRSS35 promoter were predicted and functionally validated through luciferase assays, connecting low HNF4A expression in HCC with reduced PRSS35 levels .

How might PRSS35 serve as a biomarker in cancer and other diseases?

PRSS35's potential as a disease biomarker can be explored through these methodological approaches:

• Clinical sample analysis strategy:

  • Develop standardized ELISA protocols for serum PRSS35 quantification

  • Establish reference ranges in healthy populations

  • Compare levels across disease states and stages

• Performance metrics assessment:

  Parameter	Findings for HCC
  Sensitivity	Significant reduction in HCC patients vs. controls
  Specificity	Differentiates HCC from non-malignant conditions
  Prognostic value	Higher PRSS35 correlates with longer survival
  Stage correlation	Gradual reduction with increasing HCC stage

• Multi-marker panel integration:

  • Combine PRSS35 with established biomarkers

  • Develop algorithms to enhance diagnostic accuracy

  • Validate in independent patient cohorts

• Form-specific biomarker analysis:

  • Distinguish between full-length and processed PRSS35 forms

  • Assess truncated PRSS35 forms in patient serum

  • Develop assays specific for functionally relevant forms

• Therapeutic monitoring applications:

  • Track PRSS35 levels during treatment

  • Correlate changes with treatment response

  • Evaluate potential as a predictive biomarker

Research has demonstrated that PRSS35 levels are markedly decreased in HCC lesions compared to adjacent non-cancerous tissues, with patients expressing high PRSS35 exhibiting longer survival times. Additionally, truncated PRSS35 forms show marked reduction in HCC patient serum compared to normal subjects .

What considerations are important when developing therapeutic strategies targeting the PRSS35 pathway?

Developing therapeutic strategies targeting the PRSS35 pathway requires careful consideration of these methodological aspects:

• Mechanism-based therapeutic approaches:

  • Recombinant PRSS35 administration: Supplying active protein to compensate for deficiency

  • Transcriptional activation: Enhancing endogenous PRSS35 expression through HNF4A modulation

  • Downstream effector targeting: Inhibiting CXCL2 or neutrophil extracellular trap formation

• Model system selection for preclinical evaluation:

  • Cell culture systems: Limited value due to PRSS35's microenvironment-dependent effects

  • Xenograft models: Useful for human PRSS35 studies in immunocompromised hosts

  • Immunocompetent models: Essential for studying immune microenvironment interactions

  • PRSS35-knockout mice: Valuable for pathway validation studies

• Delivery system optimization:

  • Targeted delivery to specific tissues (e.g., liver for HCC applications)

  • Protection of recombinant PRSS35 from degradation

  • Controlled release systems for sustained activity

• Combination therapy strategies:

  • Integration with conventional treatments (chemotherapy, immunotherapy)

  • Sequential treatment approaches

  • Biomarker-guided patient selection

• Safety and efficacy monitoring:

  • Development of antibodies for detecting therapeutic PRSS35

  • Distinguishing endogenous from exogenous PRSS35

  • Monitoring immune responses to PRSS35-based therapeutics

The tumor suppressive effects observed in multiple mouse models (xenograft HCC, spontaneous YAP-5SA-induced HCC, and Hepa1-6 murine HCC) suggest therapeutic potential, with the interesting observation that PRSS35 exhibits tumor suppressive effects in vivo but not in cultured cells, highlighting the importance of the tumor microenvironment in PRSS35 function .