PTPN3 Antibody, HRP conjugated

What is PTPN3 and why is it important in research?

PTPN3 (Protein Tyrosine Phosphatase Non-Receptor Type 3) is a member of the protein tyrosine phosphatase family that regulates various cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. The protein contains a C-terminal PTP domain and an N-terminal domain homologous to the band 4.1 superfamily of cytoskeletal-associated proteins. PTPN3 has gained research importance due to its involvement in multiple cellular pathways and disease mechanisms. It contains a PDZ domain that mediates protein-protein interactions, particularly with viral proteins like HBc (Hepatitis B virus core protein) through their PDZ-binding motifs (PBMs) . PTPN3 has been implicated in various cancers, including breast, lung, colorectal cancer, intrahepatic cholangiocarcinoma, and hepatocellular carcinoma . Additionally, PTPN3 functions as a tumor suppressor by enhancing TGF-β signaling and acts as an immune checkpoint in activated lymphocytes .

What are the technical specifications of the PTPN3 Antibody, HRP conjugated?

The PTPN3 Antibody, HRP conjugated, is a Rabbit Polyclonal antibody specifically targeting human Protein Tyrosine Phosphatase, Non-Receptor Type 3. The antibody is conjugated to Horseradish Peroxidase (HRP) for direct detection without requiring secondary antibodies. It has an IgG isotype and is purified by Protein G with a purity level greater than 95% . The antibody is supplied in liquid form in a buffer containing 0.01 M PBS (pH 7.4), 0.03% Proclin-300, and 50% Glycerol . For optimal results, it should be stored in aliquots at -20°C, with care taken to avoid exposure to light and repeated freeze/thaw cycles . The UniProt ID for the target protein is P26045, and the corresponding gene ID is 5774 .

What cellular processes and signaling pathways involve PTPN3?

PTPN3 participates in multiple cellular processes and signaling pathways:

• TGF-β Signaling: PTPN3 enhances TGF-β-induced transcriptional responses and functions as a tumor suppressor by boosting TGF-β signaling . Transient expression of PTPN3 markedly enhances TGF-β-induced CAGA-luc reporter activity in various cell lines, including HaCaT, A549, Huh7, and SNU449 cells . Knockdown of PTPN3 abolishes TGF-β-induced expression of endogenous p21 and PAI-1 mRNA and proteins while affecting the downregulation of c-Myc .

• T-cell Activation: PTPN3 functions as an immune checkpoint in activated lymphocytes. Its expression significantly increases during T-cell activation with IL-2 and anti-CD3 mAb . Inhibiting PTPN3 expression in activated lymphocytes augments proliferation, migration, and cytotoxicity through the phosphorylation of ZAP-70, LCK, and ERK .

• Viral Interactions: PTPN3 interacts with viral proteins through its PDZ domain. It binds to the C-terminal PDZ-binding motif of Hepatitis B virus core protein (HBc) and is targeted by high-risk human papillomavirus (HPV) types 16 and 18 through the PBM of the viral E6 oncoprotein .

• Cell Cycle Regulation: P97, a cell cycle regulator involved in membrane-related functions, is a substrate of PTPN3 .

• Adaptor Protein Interaction: PTPN3 interacts with and is regulated by adaptor protein 14-3-3 beta .

What are the validated applications for PTPN3 Antibody, HRP conjugated?

The PTPN3 Antibody, HRP conjugated, has been validated for ELISA applications according to the product specifications . While ELISA is the specifically tested application, the antibody may potentially be suitable for other immunodetection methods where HRP-conjugated antibodies are commonly employed. These could include:

• Western Blotting: For detecting PTPN3 protein expression in cell and tissue lysates. Researchers have used PTPN3 antibodies to demonstrate expression changes in various experimental settings, such as TGF-β stimulation studies .

• Immunohistochemistry (IHC): To localize PTPN3 in tissue sections, though this would require optimization and validation by the end user.

• Immunocytochemistry (ICC): For cellular localization studies of PTPN3, which could be particularly useful when investigating its interactions with viral proteins or signaling components .

• Protein Arrays: For high-throughput screening of PTPN3 expression or interactions.

The optimal dilutions/concentrations for any application should be determined by the end user for their specific experimental conditions, as indicated in the product specifications .

How should researchers optimize ELISA protocols for PTPN3 detection?

To optimize ELISA protocols for PTPN3 detection using the HRP-conjugated antibody, researchers should consider the following approach:

• Antibody Titration:

  • Prepare a dilution series of the antibody (e.g., 1:500, 1:1000, 1:2000, 1:5000, 1:10000)

  • Test these dilutions against standardized amounts of PTPN3 (recombinant protein or lysates with known expression)

  • Select the dilution providing the best signal-to-background ratio

• Sample Preparation:

  • For cell lysates: Use an appropriate lysis buffer containing protease inhibitors

  • Determine optimal protein concentration for coating (direct ELISA) or detection (sandwich ELISA)

  • Consider different lysis conditions if PTPN3 is difficult to extract

• Blocking Optimization:

  • Test different blocking reagents (BSA, non-fat dry milk, commercial blocking buffers)

  • Optimize blocking duration and temperature

• Detection System:

  • Select appropriate HRP substrate based on required sensitivity (TMB, ABTS, OPD)

  • Optimize substrate incubation time for maximum signal with minimal background

• Controls:

  • Include positive controls: Lysates from cells known to express PTPN3 (e.g., activated lymphocytes , HaCaT, A549 cells )

  • Include negative controls: Lysates from PTPN3 knockdown cells or cells naturally not expressing PTPN3

  • Include blank wells (no sample) to assess background

• Validation:

  • Confirm specificity using competitive inhibition with recombinant PTPN3

  • Compare results with alternative methods (e.g., Western blotting)

The optimal protocol will provide a linear relationship between PTPN3 concentration and signal intensity within your expected sample concentration range.

What are the best approaches for detecting PTPN3 interactions with viral proteins?

Several sophisticated approaches can be employed to study PTPN3 interactions with viral proteins, particularly the Hepatitis B virus core protein (HBc) and HPV E6 protein mentioned in the search results :

• Co-immunoprecipitation (Co-IP) with Western Blotting:

  • Immunoprecipitate PTPN3 using a non-conjugated antibody

  • Detect co-precipitated viral proteins using virus-specific antibodies

  • The HRP-conjugated PTPN3 antibody can be used for confirming PTPN3 presence

  • This approach has been used to demonstrate PTPN3 binding to HBc within capsids or as a homodimer

• Structural Analysis Complementation:

  • The crystal structure of the PDZ domain of PTPN3 in complex with the PBM of HBc has been solved

  • The HRP-conjugated antibody can verify expression levels in structural studies

• Pull-down Assays:

  • Use recombinant PTPN3-PDZ domain for pull-down assays to identify interacting viral and host proteins

  • This approach has been used to detect endogenous PBM-containing proteins that potentially interact with PTPN3

• Functional Impact Studies:

  • Investigate the effects of PTPN3 overexpression on viral infection, as demonstrated with HBV infection in HepG2 NTCP cells

  • Use the antibody to confirm PTPN3 expression levels in these functional studies

• PDZ Domain Screening:

  • High-throughput analysis of viral protein interactions using PDZ domain libraries

  • This approach identified that HBV targets the same PDZ-containing proteins as the high-risk HPV oncovirus

• Competitive Binding Assays:

  • Investigate competition between viral PBMs and endogenous cellular PBM-containing proteins for binding to PTPN3-PDZ

These approaches provide comprehensive insights into PTPN3's interactions with viral proteins and their functional significance in viral pathogenesis.

How can researchers investigate PTPN3's role as an immune checkpoint using this antibody?

To investigate PTPN3's role as an immune checkpoint in T-cell activation using the HRP-conjugated PTPN3 antibody, researchers can implement the following research strategies:

• Expression Profiling During T-cell Activation:

  • Monitor PTPN3 expression levels in T-cells at different activation stages

  • The research shows that PTPN3 expression significantly increases during lymphocyte activation with IL-2 and anti-CD3 mAb

  • Use the HRP-conjugated antibody in Western blotting or ELISA to quantify this upregulation

• Signaling Pathway Analysis:

  • Correlate PTPN3 expression levels with phosphorylation status of key T-cell signaling molecules:

    • ZAP-70 (zeta-chain-associated protein kinase 70)

    • LCK (lymphocyte-specific protein tyrosine kinase)

    • ERK (extracellular signal-regulated kinases)

  • Research has shown that inhibiting PTPN3 increases phosphorylation of these proteins

• Genetic Manipulation Studies:

  • Create PTPN3 knockdown models using siRNA or shRNA

  • Use the antibody to confirm knockdown efficiency

  • Assess functional parameters in these models:

    • T-cell proliferation

    • Migration capacity

    • Cytotoxic activity

  • Results should confirm the finding that PTPN3 inhibition significantly augments these functions

• Combinatorial Checkpoint Analysis:

  • Investigate PTPN3 in combination with other established immune checkpoints

  • Compare expression patterns and functional outcomes when modulating multiple checkpoints

• Ex Vivo Human Sample Analysis:

  • Use the antibody to measure PTPN3 levels in patient-derived T-cells

  • Correlate expression with clinical parameters or response to immunotherapy

This research approach would build upon the finding that "PTPN3 acts as an immune checkpoint in activated lymphocytes and that PTPN3 inhibitor may be a new non-antibody-based checkpoint inhibitor for cancer immunotherapy" .

What techniques can be used to correlate PTPN3 phosphatase activity with protein expression?

Correlating PTPN3 phosphatase activity with protein expression levels requires sophisticated techniques that combine detection of protein abundance with functional enzymatic assessment:

• Parallel Activity and Expression Analysis:

  • Quantify PTPN3 protein levels using the HRP-conjugated antibody in ELISA or Western blotting

  • In parallel, measure phosphatase activity using:

    • pNPP (para-nitrophenylphosphate) assay

    • Phosphotyrosine peptide dephosphorylation assays

    • Dephosphorylation of specific protein substrates

  • Plot activity as a function of protein expression

• Mutant Protein Comparative Analysis:

  • Generate expression constructs for wild-type PTPN3 and catalytically inactive mutants (e.g., C842S or D811A as mentioned in search result #3)

  • Transfect cells with these constructs

  • Detect protein expression using the antibody

  • Compare phosphatase activity in cell lysates

  • This approach distinguishes between expression levels and catalytic efficiency

• Substrate Phosphorylation Monitoring:

  • Measure phosphorylation status of known PTPN3 substrates:

    • P97, a cell cycle regulator mentioned as a PTPN3 substrate

    • Components of the TGF-β signaling pathway

    • ZAP-70, LCK, and ERK in T-cells

  • Correlate substrate phosphorylation with PTPN3 expression levels

• Dynamic Regulation Studies:

  • Track PTPN3 expression and activity following cellular stimulation

    • TGF-β treatment in epithelial cells

    • IL-2 and anti-CD3 mAb activation in lymphocytes

  • Determine if post-translational modifications affect the relationship between expression and activity

How can researchers investigate PTPN3's role in TGF-β signaling pathways?

To investigate PTPN3's role in TGF-β signaling pathways using the HRP-conjugated PTPN3 antibody, researchers can implement these evidence-based methodologies:

• Expression-Response Correlation Studies:

  • Modulate PTPN3 expression through:

    • Transient overexpression (shown to enhance TGF-β-induced CAGA-luc reporter activity)

    • siRNA knockdown (shown to decrease TGF-β-induced reporter gene activity)

  • Use the HRP-conjugated antibody to confirm and quantify expression levels

  • Measure TGF-β responses using:

    • Luciferase reporter assays (CAGA-luc)

    • Expression of endogenous target genes (PAI-1, p21)

    • Protein levels of TGF-β targets (PAI-1, p21, c-Myc)

• Molecular Mechanism Analysis:

  • Investigate PTPN3 interaction with TGF-β pathway components through:

    • Co-immunoprecipitation experiments

    • Proximity ligation assays

  • Assess phosphorylation status of TGF-β signaling molecules in the presence/absence of PTPN3

• Time-Course Studies:

  • Analyze the temporal dynamics of:

    • PTPN3 expression following TGF-β stimulation

    • TGF-β target gene activation

    • Smad phosphorylation and nuclear translocation

  • Research shows TGF-β increases PAI-1 and p21 mRNAs in a time-dependent manner, but PTPN3-deficient cells are less responsive

• Genome-Wide Analysis:

  • Conduct RNA-seq experiments in parental and PTPN3-depleted cells as mentioned in search result #3

  • Identify global TGF-β gene responses regulated by PTPN3

  • Validate key findings using qRT-PCR and protein expression analysis with the HRP-conjugated antibody

• Functional Outcome Assessment:

  • Investigate PTPN3's impact on TGF-β-mediated biological processes:

    • EMT (epithelial-mesenchymal transition)

    • Growth inhibition

    • ECM production

  • The research shows PTPN3 depletion attenuates TGF-β-mediated regulation of fibronectin, N-cadherin, and E-cadherin in fibroblasts

These approaches would build upon the finding that "PTPN3 acts as a tumor suppressor and boosts TGF-β signaling" , providing mechanistic insights into this regulatory relationship.

What strategies are recommended for simultaneous detection of PTPN3 and its interaction partners?

For sophisticated simultaneous detection of PTPN3 and its interaction partners in complex biological samples, researchers can employ these advanced methodologies:

• Sequential Immunoprecipitation and Detection:

  • Immunoprecipitate PTPN3 using a non-conjugated antibody

  • Detect co-precipitated partners using specific antibodies

  • Use the HRP-conjugated PTPN3 antibody to confirm PTPN3 presence

  • This approach has been used in studies of PTPN3 interaction with viral proteins like HBc

• PDZ Domain Screening Systems:

  • Pull-down assays with PTPN3-PDZ have been used to detect endogenous PBM-containing proteins that potentially interact with PTPN3 in cells

  • High-throughput screening of human PDZ domain libraries can identify potential cellular partners of PBM-containing proteins

  • The HRP-conjugated antibody can verify PTPN3 expression in these systems

• Proximity-Based Detection Methods:

  • Proximity Ligation Assay (PLA) allows in situ detection of protein-protein interactions

  • FRET (Förster Resonance Energy Transfer) analysis between labeled PTPN3 and potential partners

  • BiFC (Bimolecular Fluorescence Complementation) for visualizing interactions in living cells

• Multiplex Immunoassays:

  • Use different detection channels for simultaneous visualization of multiple proteins

  • Combine the HRP-conjugated PTPN3 antibody with fluorescently labeled antibodies against interaction partners

• Proteomics Approaches:

  • As mentioned in the search results, "proteomics studies on both sides by pull-down assays and screening of a human PDZ domain library" identified potential interactions

  • Mass spectrometry analysis following PTPN3 immunoprecipitation

  • Crosslinking mass spectrometry to capture transient or weak interactions

How can researchers differentiate between normal and pathological PTPN3 expression/function?

To differentiate between normal and pathological PTPN3 expression/function using the HRP-conjugated antibody, researchers should implement a comprehensive analytical framework:

• Expression Level Analysis in Normal vs. Diseased Tissues:

  • Quantify PTPN3 protein levels using the HRP-conjugated antibody in:

    • Normal tissues

    • Cancer tissues (PTPN3 has been implicated in breast, lung, colorectal cancer, intrahepatic cholangiocarcinoma, hepatocellular carcinoma)

    • Viral infection models (HBV, HPV)

  • Generate expression profiles across tissue types and disease states

• Functional Impact Assessment:

  • Compare PTPN3's impact on key signaling pathways:

    • TGF-β pathway (normal: enhances TGF-β signals as a tumor suppressor)

    • T-cell activation (normal: acts as an immune checkpoint)

    • Viral protein interactions (pathological: interactions with HBc, HPV E6)

  • Measure downstream effects on cell proliferation, migration, and survival

• Mutation and Variant Analysis:

  • Generate expression constructs for:

    • Wild-type PTPN3

    • Disease-associated mutants

    • Functional domain mutants (e.g., L232R, D811A, C842S mentioned in search result #3)

  • Use the antibody to confirm expression levels

  • Compare functional outcomes between variants

• Context-Dependent Expression:

  • Analyze PTPN3 expression dynamics in response to:

    • Growth factors (particularly TGF-β)

    • Immune activation signals (IL-2, anti-CD3 mAb)

    • Viral infection

  • Compare these responses between normal and pathological conditions

• Interaction Partner Profiling:

  • Identify differences in PTPN3 interaction partners between:

    • Normal cellular contexts

    • Disease states

    • Viral infections

  • Research has shown that "PTPN3 has been implicated in many cancers, and it has been suggested that PTPN3 mutations and HBV may exert synergistic effects in the origin of intrahepatic cholangiocarcinoma"

This multifaceted approach would help establish PTPN3 as a potential biomarker or therapeutic target in various pathological conditions.