PRSS56 Antibody

What is PRSS56 and why is it an important research target?

PRSS56 (protease, serine 56) is a trypsin-like serine protease that plays critical roles in multiple biological processes. Research significance stems from two major areas:

Ocular Development: PRSS56 is essential in eye development, particularly in the regulation of ocular axial length. Mutations in this gene cause autosomal recessive posterior microphthalmos, a condition characterized by severe reduction in ocular axial length and extreme hyperopia. It also contributes to angle-closure glaucoma (ACG) pathogenesis .

Cancer Biology: PRSS56 has been identified as a novel cancer-testis (CT) antigen that is frequently overexpressed in various cancers, especially in gastrointestinal cancers. It promotes cancer progression through activation of the PI3K-AKT signaling pathway .

These dual functions make PRSS56 antibodies valuable tools for research across both ophthalmology and oncology fields.

What are the most reliable methods for detecting PRSS56 expression in different tissue samples?

Detection of PRSS56 requires specific methodological considerations due to its varied expression patterns. Based on current research approaches:

Western Blotting (WB): This remains the gold standard for quantitative detection. Most commercial antibodies are validated for WB applications with a predicted protein size of approximately 63 kDa . For optimal results, tissue lysates should be prepared with protease inhibitors to prevent degradation of this protease.

Immunohistochemistry (IHC): Both paraffin-embedded (IHC-P) and frozen sections (IHC-F) techniques are viable. Recommended dilutions typically range from 1:100-1:500 for frozen sections and 1:200-1:400 for paraffin sections . Antigen retrieval methods significantly impact detection sensitivity.

Immunofluorescence (IF): Validated protocols exist for both tissue sections and cell cultures with recommended dilutions of 1:50-1:200 .

qRT-PCR: For detection at the mRNA level, especially valuable when studying methylation-dependent expression changes. Research has shown significant sensitivity in detecting PRSS56 upregulation after DNA methyltransferase inhibitor treatment .

The approach selection should be guided by whether protein localization or quantification is the primary objective.

How should researchers validate PRSS56 antibodies for experimental applications?

Validation of PRSS56 antibodies requires a multi-faceted approach:

Positive Controls:

• For cancer research: Gastric cancer (GC) and colorectal cancer (CRC) cell lines, particularly after treatment with DNA methyltransferase inhibitors (e.g., 5-AZA-CdR), which significantly upregulate PRSS56 expression .

• For ocular research: Testis tissue shows high endogenous expression, while normal retinal tissue shows lower but detectable expression .

Negative Controls: Include brain cancer tissue which typically shows minimal expression, or use siRNA knockdown in expressing cell lines .

Epitope Considerations: Most validated antibodies target the C-terminal region of PRSS56. For example, antibodies using immunogens within "SRAAGTRFPKRRPEPRGEANGCPGLEPLRQKLAALQGAHAWILQVPSEHL" sequence have demonstrated reliability . This is particularly important as mutations affecting the C-terminal region have been associated with ocular phenotypes .

Species Reactivity Verification: While many antibodies claim cross-reactivity, direct validation is necessary. Most commercial antibodies show confirmed reactivity to human PRSS56, with predicted cross-reactivity to mouse, dog, pig, and rabbit variants based on immunogen sequence homology .

A comprehensive validation approach would include both genetic manipulation (overexpression/knockdown) and comparison across multiple antibodies targeting different epitopes.

What are the optimal storage and handling conditions for PRSS56 antibodies?

Optimal handling of PRSS56 antibodies requires attention to protein stability factors:

Storage Temperature: Most purified antibodies should be stored at -20°C for long-term stability. Many commercial PRSS56 antibodies include glycerol in the buffer to prevent freeze-thaw damage .

Buffer Composition: Typical storage buffers include:

• PBS (1x) with 0.09% sodium azide and 2% sucrose

• TBS (0.01M, pH 7.4) with 1% BSA, 0.02% Proclin300, and 50% Glycerol

Freeze-Thaw Cycles: Minimize repeated freeze-thaw cycles as they significantly reduce antibody activity. Aliquoting upon receipt is recommended .

Working Solutions: For diluted working solutions, storage at 4°C is generally acceptable for up to one week. For longer periods, re-freezing small aliquots is preferable.

Shipping Considerations: Most suppliers ship the antibodies under cool pack conditions, maintaining 4°C throughout transport . Upon receipt, immediate transfer to -20°C storage is recommended.

Following these guidelines ensures maintained specificity and sensitivity for experimental applications.

How can PRSS56 antibodies be utilized to investigate DNA methylation-dependent gene regulation?

PRSS56 represents an excellent model for studying epigenetic regulation through DNA methylation. Research methodologies using PRSS56 antibodies in this context include:

ChIP-Seq Combined with Bisulfite Sequencing: This approach allows correlation between protein binding and methylation status. PRSS56 expression has been shown to correlate negatively with promoter methylation and positively with gene body methylation .

Methylation-Specific Antibody Studies: Researchers can treat cells with DNA methyltransferase inhibitors (5-AZA-CdR) and track PRSS56 expression changes using validated antibodies. Studies have demonstrated that 5-AZA-CdR treatment significantly decreased DNA methylation at the CpG site cg10242318 in the PRSS56 promoter, resulting in activation of PRSS56 expression .

Time-Course Experiments: Track the activation of PRSS56 following demethylation treatments. Research has shown 5-Aza-CdR treatment in HCT116 and AGS cell lines resulted in significant upregulation (log2 FC > 2) of PRSS56, similar to known cancer-testis antigens like SPANXB1, DAZL, and TKTL1 .

Targeted Methylation Analysis: Using antibodies against methylated DNA in conjunction with PRSS56 antibodies can identify specific regulatory regions. The GSE29290 dataset analysis revealed that DNMT1/DNMT3B knockout significantly decreased PRSS56 promoter DNA methylation .

These approaches provide powerful tools for understanding the epigenetic mechanisms controlling PRSS56 expression in both normal development and pathological conditions.

What methodologies are recommended for studying PRSS56's role in oncogenic signaling pathways?

Investigation of PRSS56's role in oncogenic signaling, particularly in the PI3K/AKT pathway, requires sophisticated experimental approaches:

Phosphorylation-State Specific Co-Immunoprecipitation: Using PRSS56 antibodies in conjunction with phospho-specific antibodies against AKT pathway components. Research has demonstrated that PRSS56 overexpression significantly activates the PI3K/AKT signaling pathway in gastric and colorectal cancer cells .

Gain/Loss-of-Function Studies: Creating stable cell lines with altered PRSS56 expression followed by pathway analysis:

• Lentiviral-mediated PRSS56 overexpression in HCT116 and AGS cell lines has been validated to accelerate cell growth and enhance migration/invasion .

• PRSS56 knockdown models demonstrated reduced activation of PI3K/AKT pathway components.

Pharmacological Inhibitor Rescue Experiments: Combining PRSS56 overexpression with pathway inhibitors. Research has shown that the promoting effect of PRSS56 overexpression on GC and CRC cell proliferation can be partially or completely eliminated after incubation with the PI3K inhibitor LY294002 .

Transcriptome Analysis: RNA-seq comparisons between PRSS56-high and PRSS56-low expressing cells. Analysis of TCGA_STAD cohort data identified significant differences in gene expression patterns, with PRSS56 overexpression involved in activating PI3K/AKT and Wnt/beta-catenin signaling pathways .

These methodologies provide comprehensive approaches to characterize PRSS56's oncogenic mechanisms, potentially identifying novel therapeutic targets.

How can PRSS56 antibodies be utilized in ocular research to study eye development disorders?

PRSS56 antibodies offer specialized applications for investigating ocular development disorders:

Retinal Layer-Specific Detection: Using immunohistochemistry to localize PRSS56 expression in retinal structures. Studies have shown PRSS56 expression predominantly in the inner nuclear layer (INL) of the retina, a region where Müller glia cell bodies are located .

Developmental Timeline Studies: Tracking PRSS56 expression changes during critical periods of eye development. Research has demonstrated progressive upregulation of PRSS56 mRNA from P15 to P60 in mouse models, suggesting temporally regulated functions .

Disease Model Analysis: Several mouse models have been developed that mimic human ocular phenotypes:

• The Grm4 mutation disrupts a splice donor site in Prss56 exon 11, resulting in intron retention and premature stop codon .

• Conditional gene targeting approaches allowing temporal control of Prss56 inactivation have shown alterations in axial length regulation .

Genetic Interaction Studies: Investigating PRSS56 in the context of other ocular development genes. Research has demonstrated that:

• PRSS56 interacts with MFRP (membrane frizzled-related protein) in ocular size determination .

• Loss of either PRSS56 or MFRP prevents excessive ocular elongation in early-onset myopia models .

These methodological approaches provide valuable insights into microphthalmos, angle-closure glaucoma, and other ocular size disorders.

What considerations should be made when using PRSS56 antibodies for cancer immunotherapy research?

As a cancer-testis antigen with high immunogenicity, PRSS56 represents a promising target for cancer immunotherapy research:

Epitope Selection for Vaccine Development: When designing cancer vaccines targeting PRSS56, antibodies can help identify immunogenic epitopes. Research has identified PRSS56 as selectively expressed in early spermatids of normal testis while being widely upregulated in various cancers .

Tumor Microenvironment Analysis: Using multiplex immunofluorescence with PRSS56 antibodies alongside immune cell markers. KEGG pathway analysis has shown that PRSS56 is negatively associated with complement activation, antigen and immunoglobulin receptor binding .

Patient Selection Biomarker Development: PRSS56 expression varies across cancer types and patients:

• It is frequently overexpressed in gastrointestinal cancers

• Expression correlates with promoter DNA methylation status

Cross-Reactivity Assessment: Due to PRSS56's normal expression in testis, careful analysis of antibody cross-reactivity with normal tissues is essential. Studies have confirmed that PRSS56 shows restricted expression patterns in normal tissues, primarily in testis with minimal expression in other tissues .

These approaches facilitate the development of targeted immunotherapies while minimizing off-target effects in normal tissues.

What are the most common issues encountered when using PRSS56 antibodies, and how can they be resolved?

Research using PRSS56 antibodies may encounter several technical challenges:

Low Signal Intensity:

• Problem: PRSS56 is often expressed at low levels in normal tissues.

• Solution: Employ signal amplification systems like tyramide signal amplification (TSA). Additionally, treating cells with DNA methyltransferase inhibitors (5-AZA-CdR) can significantly increase PRSS56 expression for positive controls .

Background Staining:

• Problem: Non-specific binding, especially in tissues with high protease activity.

• Solution: Include proper blocking protocols (3-5% BSA or normal serum). For tissues with high endogenous protease activity, additional protease inhibitors during sample preparation are recommended.

Inconsistent Results Between Applications:

• Problem: An antibody that works for Western blotting may not work for immunohistochemistry.

• Solution: Verify application-specific validation. Many PRSS56 antibodies are specifically validated for particular applications with different optimal dilutions (e.g., 1:500-1000 for ELISA vs. 1:50-200 for immunofluorescence) .

Cross-reactivity Issues:

• Problem: Potential cross-reactivity with other serine proteases.

• Solution: Validate specificity using PRSS56 knockout models or siRNA knockdown approaches. When selecting antibodies, prioritize those targeting unique regions not conserved among other serine proteases.

These systematic troubleshooting approaches can significantly improve experimental outcomes when working with PRSS56 antibodies.

How can researchers optimize immunohistochemical protocols for detecting PRSS56 in different cancer types?

Optimizing immunohistochemical detection of PRSS56 across diverse cancer tissues requires tailored approaches:

Antigen Retrieval Optimization:

• Gastrointestinal Cancers: Heat-induced epitope retrieval in citrate buffer (pH 6.0) has shown optimal results for PRSS56 detection in gastric and colorectal cancers .

• Different cancer types may require testing multiple retrieval buffers (citrate pH 6.0, EDTA pH 8.0, or Tris-EDTA pH 9.0).

Signal Amplification Strategies:

• Tyramide signal amplification has been effective for detecting low-abundance PRSS56.

• Polymeric detection systems show higher sensitivity than ABC methods for PRSS56 detection.

Counterstaining Considerations:

• When studying PRSS56 in relation to cell morphology, hematoxylin counterstaining time may need adjustment as excessive counterstaining can mask subtle PRSS56 signals.

Multiplex Immunostaining Approaches:

• For correlating PRSS56 with pathway markers (e.g., phospho-AKT), sequential multiplex protocols have proven effective.

• Research has shown co-localization of PRSS56 with PI3K/AKT pathway components in gastric cancer tissues .

Control Tissue Selection:

• Positive Controls: Testis tissue for normal expression; gastric and colorectal cancer tissues for pathological expression.

• Negative Controls: Brain cancer tissue typically shows minimal expression .

These optimizations significantly improve detection sensitivity and specificity across diverse tumor types, enabling more accurate assessment of PRSS56's role in cancer progression.

How can PRSS56 antibodies be used to investigate the relationship between ocular development and cancer biology?

The dual role of PRSS56 in both ocular development and cancer biology offers unique research opportunities:

Comparative Immunoprofiling:

• Using the same validated antibodies to compare PRSS56 expression patterns in ocular tissues versus cancer tissues.

• Research has identified that while PRSS56 regulates ocular size through specific developmental mechanisms, its activation in cancer involves distinct pathways including PI3K/AKT signaling .

Signaling Pathway Crosstalk Analysis:

• Eye development research has shown PRSS56 interacts with MFRP and influences ADAMTS19 expression .

• Cancer research has demonstrated PRSS56 activates PI3K/AKT signaling .

• Comparative antibody-based studies can identify shared versus tissue-specific interaction partners.

Methylation Regulation Comparisons:

• PRSS56 expression is regulated by DNA methylation in cancer contexts .

• Investigation of whether similar epigenetic mechanisms control PRSS56 during ocular development using methylation-specific antibodies alongside PRSS56 detection.

Therapeutic Target Evaluation:

• Antibody-based screening approaches to identify compounds that selectively modulate PRSS56 in cancer cells without affecting normal ocular development pathways.

This cross-disciplinary approach may reveal evolutionary conservation of PRSS56 functions and identify novel therapeutic strategies with reduced side effects.

What are the methodological considerations for using PRSS56 antibodies in research on serine protease activity regulation?

As a trypsin-like serine protease, investigating PRSS56's enzymatic functions requires specialized methodological approaches:

Activity-Based Probes:

• Combining PRSS56 antibodies with activity-based serine protease probes can differentiate between active and inactive enzyme forms.

• This approach allows identification of contexts where PRSS56 protein is present but enzymatically inactive.

Substrate Identification Protocols:

• Immunoprecipitation with PRSS56 antibodies followed by mass spectrometry can identify potential substrates.

• Validation requires in vitro cleavage assays with recombinant PRSS56 and candidate substrates.

Inhibitor Screening Methodologies:

• Antibody-based detection of PRSS56 can confirm protein presence in inhibitor screens focusing on enzymatic activity.

• Research has utilized PI3K inhibitor LY294002 to study downstream effects of PRSS56 in cancer cells .

Post-Translational Modification Analysis:

• Phospho-specific antibodies used alongside PRSS56 antibodies can identify regulatory modifications.

• Western blots under non-reducing versus reducing conditions can reveal structural changes affecting activity.

Zymography Techniques:

• Gelatin or casein zymography combined with PRSS56 antibody confirmation can verify proteolytic activity.

• This approach is particularly valuable when studying PRSS56's potential role in extracellular matrix remodeling.