PTPN4 Antibody

What is PTPN4 and what is its primary function in cellular processes?

PTPN4, also known as PTP-MEG1 or MEG, belongs to the PTPH1-family of protein tyrosine phosphatases. It contains highly homologous N-terminal FERM-, central PDZ-, and C-terminal catalytic-domains . PTPN4 functions primarily as a regulator of T cell receptor signaling processes by controlling protein tyrosine phosphorylation events. Studies have shown that PTPN4 can complex and dephosphorylate the ITAMs of the TCR ζ subunit, suggesting its role as a negative regulator of immune signaling . Additionally, PTPN4 plays a critical role in neurodevelopment, as evidenced by recent identification of germline variants that result in neurodevelopmental abnormalities .

How can I distinguish between PTPN4 and other protein tyrosine phosphatases?

PTPN4 belongs to the classical protein tyrosine phosphatase family but has distinctive structural and functional characteristics that differentiate it from related phosphatases like PTPN1, PTPN2, and PTPN6. While many PTPs share similar catalytic domains, PTPN4 is distinguished by its specific domain architecture including the PDZ domain which has been shown to regulate its phosphatase activity . When designing experiments, researchers should use antibodies that target unique epitopes of PTPN4 to avoid cross-reactivity with other phosphatases. Validation techniques such as Western blotting against recombinant proteins or using knockout cell lines can confirm specificity of anti-PTPN4 antibodies.

What are the primary tissues and cell types where PTPN4 is expressed?

PTPN4 has been detected in multiple tissues, with expression studies showing its presence in thymus, spleen, and testes. According to research, PTPN4 can be cloned from RNA isolated from murine thymic tissue and is expressed in T cells where it regulates TCR signaling . For tissue-specific expression analysis, researchers have successfully isolated RNA using Trizol extraction procedures and performed RT-PCR to detect PTPN4 transcripts . Different tissues may exhibit varying levels of PTPN4 expression, making it important to optimize antibody concentrations when analyzing specific cell types or tissues.

What are the optimal conditions for using anti-PTPN4 antibodies in Western blotting?

For Western blotting applications with anti-PTPN4 antibodies, researchers should consider the following protocol:

• Sample preparation: Prepare cell or tissue lysates using a buffer containing phosphatase inhibitors to preserve the phosphorylation state.

• Protein separation: Use 8-10% SDS-PAGE gels for optimal resolution of PTPN4 (approximately 105 kDa).

• Transfer conditions: Transfer to PVDF membranes at 100V for 90 minutes in standard transfer buffer.

• Blocking: Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute anti-PTPN4 antibodies (such as those produced in rabbit) at 1:1000 in blocking buffer and incubate overnight at 4°C .

• Detection: Use appropriate secondary antibodies and develop using chemiluminescence.

Validation controls should include known positive samples (e.g., thymus or spleen tissue) and negative controls (tissues with minimal PTPN4 expression).

How can I optimize immunohistochemistry protocols for PTPN4 detection in tissue sections?

For optimal immunohistochemical detection of PTPN4 in tissue sections:

• Fixation: Use 4% paraformaldehyde for 24 hours followed by paraffin embedding.

• Section thickness: Prepare 4-5 μm sections for optimal antibody penetration.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes.

• Blocking: Block endogenous peroxidase with 3% H₂O₂ and non-specific binding with 5% normal serum.

• Primary antibody: Apply affinity-isolated anti-PTPN4 antibodies (such as Prestige Antibodies® from Atlas Antibodies) at 1:100 dilution overnight at 4°C .

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

• Counterstaining: Counterstain with hematoxylin for nuclear visualization.

Tissue-specific optimization may be required depending on the expression level of PTPN4 in your target tissue.

What are the recommended approaches for validating PTPN4 antibody specificity?

To ensure antibody specificity for PTPN4:

• Western blot validation: Compare band patterns with predicted molecular weight (~105 kDa for full-length PTPN4).

• Competing peptide assay: Pre-incubate antibody with immunizing peptide to confirm specific binding.

• Immunoprecipitation followed by mass spectrometry: Confirm pulled-down protein identity.

• Genetic approaches: Use PTPN4 knockout or knockdown samples as negative controls. PTPN4-deficient mice have been developed that can serve as important controls .

• Cross-reactivity testing: Test antibody against closely related phosphatases (particularly other PTPH1-family members).

• Cell/tissue panel: Validate antibody performance across multiple cell types with known differential expression of PTPN4.

Researchers should document all validation steps and include appropriate controls in their experimental design to ensure result reproducibility.

How can PTPN4 antibodies be utilized to study neurodevelopmental disorders associated with PTPN4 mutations?

Recent studies have identified germline variants in PTPN4 that result in aberrant neurodevelopment . To investigate these disorders:

• Mutation-specific antibodies: Develop or utilize antibodies that can distinguish between wild-type and mutant PTPN4 (e.g., p.Arg808His variant) .

• Immunohistochemistry of brain tissue: Compare PTPN4 expression patterns in normal versus affected brain regions.

• Co-immunoprecipitation studies: Use anti-PTPN4 antibodies to identify altered protein interactions in mutant versus wild-type settings.

• Phosphorylation state analysis: Employ anti-PTPN4 antibodies alongside phospho-specific antibodies to measure changes in substrate phosphorylation.

• Cellular localization studies: Use immunofluorescence with anti-PTPN4 antibodies to track potential mislocalization of mutant proteins.

When studying neurodevelopmental disorders, it's essential to combine antibody-based approaches with functional assays that measure phosphatase activity and downstream signaling effects.

What strategies can be employed to study the regulatory role of PTPN4 in T cell signaling pathways?

To investigate PTPN4's role in T cell signaling:

• TCR ζ-chain interaction studies: Use co-immunoprecipitation with anti-PTPN4 antibodies to pull down TCR complexes and analyze interactions .

• Substrate-trapping approaches: Utilize catalytically inactive PTPN4 (D to A mutation) to trap substrates, followed by immunoprecipitation and identification .

• NF-κB and AP-1 activation assays: Measure the effect of PTPN4 modulation on downstream transcription factors using luciferase reporter assays as described in previous studies .

• Phospho-flow cytometry: Combine anti-PTPN4 antibodies with phospho-specific antibodies to monitor signaling dynamics at single-cell resolution.

• Proximity ligation assays: Detect in situ interactions between PTPN4 and potential binding partners or substrates.

These approaches can help elucidate how PTPN4 regulates T cell activation, differentiation, and function in normal and pathological conditions.

How does the PDZ domain of PTPN4 regulate its phosphatase activity and how can this be studied?

Research has demonstrated that the PDZ domain of PTPN4 inhibits its phosphatase activity . To study this regulatory mechanism:

• Domain-specific antibodies: Develop antibodies targeting the PDZ domain specifically to study its conformational changes.

• In vitro phosphatase assays: Compare the activity of full-length PTPN4 versus truncated versions lacking the PDZ domain.

• Binding partner identification: Use the PDZ domain as bait in pull-down experiments followed by mass spectrometry.

• Conformational studies: Employ limited proteolysis combined with antibody epitope mapping to detect structural changes upon PDZ domain binding.

• FRET-based sensors: Design sensors that can detect interactions between the PDZ and phosphatase domains using fluorescently-labeled antibodies.

Understanding this regulatory mechanism could lead to therapeutic strategies targeting PTPN4 in disorders where its activity is dysregulated.

What are common issues when using PTPN4 antibodies and how can they be resolved?

Common challenges with PTPN4 antibodies include:

When troubleshooting, consider validating your antibody using alternative techniques and multiple positive control samples.

How can I analyze PTPN4 expression data in relation to disease states?

For analyzing PTPN4 expression in disease contexts:

• Quantitative analysis: Use image analysis software to quantify immunohistochemistry or Western blot signals, normalizing to appropriate housekeeping proteins or reference tissues.

• Statistical approaches: Apply appropriate statistical tests (t-tests for two-group comparisons, ANOVA for multiple groups) with correction for multiple testing when analyzing expression across disease states.

• Correlation studies: Perform correlation analyses between PTPN4 expression levels and clinical parameters or disease severity.

• Meta-analysis: Compare your findings with publicly available expression datasets from sources like The Cancer Genome Atlas or GEO.

• Integrative analysis: Combine antibody-based expression data with genomic data about PTPN4 variants or mutations to identify genotype-phenotype correlations.

Particular attention should be given to neurodevelopmental disorders and immunological conditions where PTPN4 has demonstrated relevance .

What considerations are important when using PTPN4 antibodies for studying the effects of PTPN4 germline variants?

When investigating PTPN4 germline variants:

• Variant-specific detection: Determine whether your antibody can detect the variant of interest or if it binds to a region affected by the mutation.

• Expression level analysis: Quantify whether the variant affects protein stability or expression using calibrated Western blots.

• Subcellular localization: Use immunofluorescence to determine if variants alter the cellular distribution of PTPN4.

• Functional correlation: Combine antibody detection with functional assays to correlate protein presence with activity.

• Tissue-specific effects: Compare variant effects across multiple tissues, particularly focusing on brain and immune tissues.

Recent studies have identified PTPN4 germline variants in individuals with growth and cognitive abnormalities , making these analyses particularly relevant for neurodevelopmental research.

How can PTPN4 antibodies be used in studying the potential role of PTPN4 in cancer?

Recent research suggests PTPN4 may play a role in cancer development:

• Tumor tissue microarrays: Use anti-PTPN4 antibodies for high-throughput screening of PTPN4 expression across tumor types.

• STAT3 signaling analysis: Investigate the relationship between PTPN4 loss and STAT3 activation in rectal cancer, as loss of PTPN4 has been shown to activate STAT3 to promote tumor growth .

• Prognostic correlation: Correlate PTPN4 expression levels with patient outcomes using immunohistochemistry on tumor samples.

• Epigenetic regulation: Investigate PTPN4 in regions affected by long-range epigenetic silencing, particularly at 2q14.2 in colorectal cancers .

• Therapeutic response prediction: Examine whether PTPN4 expression levels correlate with response to targeted therapies.

Combining antibody-based detection with functional studies will be crucial to elucidate PTPN4's role in cancer progression and its potential as a biomarker.

What technological advances are improving the sensitivity and specificity of PTPN4 detection methods?

Recent technological advances for enhanced PTPN4 detection include:

• Monoclonal antibody development: New mouse monoclonal antibodies offer improved specificity compared to polyclonal alternatives .

• Proximity-based assays: Techniques like proximity ligation assay (PLA) enable detection of PTPN4 interactions with higher sensitivity.

• Multiplex immunofluorescence: Simultaneous detection of PTPN4 and its interaction partners or substrates.

• Super-resolution microscopy: Improved visualization of PTPN4 subcellular localization beyond the diffraction limit.

• Mass cytometry (CyTOF): Metal-conjugated antibodies for high-dimensional analysis of PTPN4 in heterogeneous cell populations.

• Nanobody development: Smaller antibody fragments for improved tissue penetration and reduced background.

Researchers should evaluate which of these advanced technologies best suits their specific experimental questions regarding PTPN4 function.