PTPN13 Antibody, HRP conjugated

What is PTPN13 and what cellular functions does it regulate?

PTPN13 (Protein Tyrosine Phosphatase, Non-Receptor Type 13) is a large protein tyrosine phosphatase that plays critical roles in multiple cellular processes. It functions primarily as a negative regulator of FAS-induced apoptosis and NGFR-mediated pro-apoptotic signaling pathways . The protein contains multiple functional domains, including:

• A PTP catalytic domain at the C-terminus

• A band 4.1 superfamily domain associated with cytoskeletal proteins

• Five PDZ domains involved in protein-protein interactions

• A leucine zipper motif

PTPN13 regulates phosphoinositide 3-kinase (PI3K) signaling through dephosphorylation of PIK3R2 and interacts with the Fas receptor and I-kappa-B-alpha, suggesting its important role in Fas-mediated programmed cell death . Recent studies have also demonstrated its involvement in regulating cell motility and invasiveness in cancer cell models such as MDA-MB-231 breast cancer cells .

What epitopes are targeted by commercially available PTPN13 antibodies?

Commercial PTPN13 antibodies target various epitopes depending on the immunogen used in their production:

Researchers should select antibodies targeting epitopes relevant to their experimental needs, considering whether functional domains or specific regions are critical to their research questions.

How does species reactivity vary among PTPN13 antibodies, and what validation should researchers perform?

Available PTPN13 antibodies demonstrate variable cross-reactivity across species:

• Human-specific: BioCat antibody and Biomatik antibody

• Multi-species (Human, Mouse, Rat): Bioss antibody and ARP antibody

When using these antibodies across species, researchers should:

• Perform sequence alignment of the target epitope across species to predict potential cross-reactivity

• Include positive controls from each species to validate cross-reactivity

• Validate antibody specificity using knockout/knockdown models when available

• Compare staining patterns with published literature for consistency

• Test multiple antibody dilutions to optimize signal-to-noise ratio in each species

Reactivity claims should be experimentally validated in your specific experimental system before proceeding with full-scale studies .

What are the optimal protocols for Western blot analysis using PTPN13 HRP-conjugated antibodies?

When performing Western blot analysis with PTPN13 HRP-conjugated antibodies, researchers should follow this optimized protocol:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors

  • Heat samples at 95°C for 5 minutes in reducing conditions

  • Load 30-50μg of total protein per lane

• Gel electrophoresis:

  • Use 4-8% gradient gels due to the large size of PTPN13 (~260 kDa)

  • Run at 100V until the dye front reaches the bottom

• Transfer:

  • Perform wet transfer to PVDF membrane at 30V overnight at 4°C

  • Use transfer buffer containing 20% methanol and 0.1% SDS

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Dilute HRP-conjugated PTPN13 antibody according to manufacturer recommendations:

    • Typically 0.2-2μg/mL or 1:250-2500 dilution

  • Incubate overnight at 4°C

• Detection:

  • Wash 4 times with TBST, 5 minutes each

  • Develop using enhanced chemiluminescence substrate

  • Expect a band at approximately 260 kDa

The HRP conjugation eliminates the need for secondary antibody incubation, reducing background and cross-reactivity issues .

How should PTPN13 HRP-conjugated antibodies be optimized for immunohistochemistry applications?

For optimal immunohistochemistry results with PTPN13 HRP-conjugated antibodies:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin following standard protocols

  • Cut 4-6μm sections on positively charged slides

• Antigen retrieval optimization:

  • Test both heat-induced epitope retrieval methods:

    • Citrate buffer (pH 6.0) for 20 minutes

    • EDTA buffer (pH 9.0) for 20 minutes

  • Select the method providing optimal signal-to-noise ratio

• Blocking and antibody incubation:

  • Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

  • Block non-specific binding with 5% normal serum for 1 hour

  • Apply PTPN13 HRP-conjugated antibody at recommended dilution:

    • Starting range: 1:100-500 for IHC-P applications

    • For precise optimization: test 5-20μg/mL concentration range

  • Incubate overnight at 4°C in a humidified chamber

• Development and counterstaining:

  • Develop with DAB substrate for 2-5 minutes (monitor microscopically)

  • Counterstain with hematoxylin for 30 seconds

  • Dehydrate, clear, and mount with permanent mounting medium

Always include positive and negative controls to validate staining specificity .

What controls are essential when validating PTPN13 antibody specificity?

Proper validation of PTPN13 antibody specificity requires multiple controls:

• Positive controls:

  • Cell lines with confirmed PTPN13 expression (e.g., HeLa cells)

  • Tissues known to express PTPN13 (e.g., epithelial tissues)

  • Recombinant PTPN13 protein (full-length or epitope-specific)

• Negative controls:

  • PTPN13 knockout/knockdown samples

  • Isotype control antibody at the same concentration

  • Primary antibody omission

  • Tissues known to lack PTPN13 expression

• Specificity validation:

  • Peptide competition assays using the immunizing peptide

  • Western blot showing a single band at expected molecular weight (~260 kDa)

  • Comparing staining patterns with multiple antibodies targeting different PTPN13 epitopes

  • Correlation of protein expression with mRNA levels using qPCR

• Reproducibility assessment:

  • Test antibody performance across multiple lots

  • Evaluate consistency across different sample preparation methods

  • Document batch-to-batch variation in sensitivity and specificity

What are common causes of high background when using HRP-conjugated PTPN13 antibodies, and how can they be mitigated?

High background is a common challenge with HRP-conjugated antibodies. Specific to PTPN13 antibodies, researchers may encounter:

For Western blots specifically, using freshly prepared PVDF membranes and ensuring thorough blocking can significantly reduce background when detecting PTPN13, which has been shown to produce cleaner results in HeLa cell lysates .

How can researchers address weak or absent signals when detecting PTPN13?

When PTPN13 detection yields weak or no signal, consider these methodological approaches:

• Protein extraction optimization:

  • Use lysis buffers containing both ionic and non-ionic detergents

  • Add phosphatase inhibitors to prevent epitope dephosphorylation

  • Avoid multiple freeze-thaw cycles of protein samples

• Antibody-specific considerations:

  • Verify antibody functionality with positive control samples

  • Test alternative antibodies targeting different PTPN13 epitopes

  • Consider antibody concentration (5-20μg/mL for IHC/ICC applications)

• Application-specific modifications:

  • For Western blot: use longer exposure times, higher protein loading (50-100μg)

  • For ELISA: reduce washing stringency, increase antibody concentration (1:250-500)

  • For IHC: optimize antigen retrieval, extend antibody incubation to 48 hours at 4°C

• Technical adjustments:

  • Ensure target protein isn't being lost during processing steps

  • Verify transfer efficiency for large proteins like PTPN13 (~260 kDa)

  • Add 0.1% SDS to transfer buffer to improve transfer of large proteins

• Expression analysis:

  • Confirm PTPN13 mRNA expression in your samples via RT-PCR

  • Consider protein degradation issues if mRNA is detected but protein is not

How can PTPN13 antibodies be employed to investigate its role in Fas-mediated apoptosis pathways?

PTPN13 plays a critical role in regulating Fas-mediated apoptosis through its interaction with Fas receptor. To investigate this function:

• Co-immunoprecipitation studies:

  • Use PTPN13 antibodies for immunoprecipitation (working dilution: optimize starting at 1:250)

  • Probe for Fas receptor in immunoprecipitates

  • Analyze phosphorylation status of the Fas receptor in the presence/absence of PTPN13

• Phosphatase activity assays:

  • Immunoprecipitate PTPN13 using specific antibodies

  • Measure phosphatase activity against synthetic phosphopeptides mimicking Fas receptor phosphorylation sites

  • Compare activity of wild-type vs. catalytically inactive PTPN13

• Subcellular localization:

  • Perform immunocytochemistry (5-20μg/mL) to track PTPN13 translocation following Fas activation

  • Use confocal microscopy to visualize co-localization with Fas receptor

  • Quantify co-localization coefficients before and after apoptotic stimuli

• Functional assays:

  • Monitor apoptosis markers (caspase activation, PARP cleavage) in cells with modified PTPN13 expression

  • Compare wild-type and catalytically inactive PTPN13 expression on apoptotic responses

  • Create a time-course of PTPN13-Fas interaction during apoptosis progression

What experimental approaches are most effective for studying PTPN13's role in cell motility and invasiveness?

Recent research has implicated PTPN13 in regulating cell motility and invasiveness, particularly in cancer models . Effective experimental designs include:

• Expression modulation studies:

  • Compare wild-type PTPN13 with catalytically inactive mutants (CS)

  • Establish stable cell lines with varied PTPN13 expression levels

  • Monitor effects on cell growth using MTS assays

• Directional migration assays:

  • Perform wound healing assays to assess directional migration

  • Document migration at multiple timepoints (0h, 9h, etc.)

  • Quantify wound closure rates between control and PTPN13-modulated cells

• Invasiveness assessment:

  • Use transwell invasion assays with Matrigel coating

  • Analyze matrix metalloproteinase expression and activity

  • Correlate invasive capacity with PTPN13 phosphatase activity

• Cytoskeletal dynamics:

  • Investigate PTPN13's interaction with GTPase-activating proteins

  • Monitor Rho signaling pathway activation

  • Analyze cytoskeletal reorganization using fluorescent microscopy

• In vivo models:

  • Develop xenograft models with PTPN13-modulated cancer cells

  • Track metastatic potential and invasiveness

  • Correlate findings with human tumor samples using IHC staining protocols (1:25-100 dilution)

How can researchers design experiments to investigate PTPN13's role in phosphoinositide 3-kinase signaling?

PTPN13 has been shown to regulate phosphoinositide 3-kinase (PI3K) signaling through dephosphorylation of PIK3R2 . To investigate this role:

• Substrate validation:

  • Perform in vitro dephosphorylation assays with recombinant PTPN13 and phosphorylated PIK3R2

  • Use phospho-specific antibodies to monitor PIK3R2 phosphorylation states

  • Compare wild-type PTPN13 with phosphatase-dead mutants

• Signaling pathway analysis:

  • Monitor Akt phosphorylation (downstream of PI3K) in cells with modulated PTPN13 expression

  • Analyze PI3K activity using PIP3 detection assays

  • Investigate feedback regulation mechanisms between PI3K and PTPN13

• Domain mapping:

  • Create PTPN13 truncation mutants to identify domains required for PIK3R2 interaction

  • Perform co-immunoprecipitation studies using antibodies targeting different PTPN13 domains

  • Use proximity ligation assays to visualize PTPN13-PIK3R2 interactions in situ

• Functional consequences:

  • Assess cellular outcomes (proliferation, survival, metabolism) influenced by PTPN13-mediated PI3K regulation

  • Investigate resistance to PI3K inhibitors in cells with altered PTPN13 expression

  • Analyze crosstalk between PTPN13-PI3K and other signaling pathways

What methods provide the most reliable quantification of PTPN13 expression across different experimental conditions?

For reliable quantification of PTPN13 expression:

• Western blot quantification:

  • Use HRP-conjugated PTPN13 antibodies at optimized dilutions (0.2-2μg/mL)

  • Include loading controls (β-actin, GAPDH) for normalization

  • Utilize standard curves with recombinant PTPN13 for absolute quantification

  • Employ digital image analysis software for densitometry

  • Perform technical triplicates and biological replicates

• ELISA-based quantification:

  • Develop sandwich ELISA using capture and detection antibodies against different PTPN13 epitopes

  • Use HRP-conjugated detection antibodies at 1:500-1:1000 dilution

  • Create standard curves using recombinant PTPN13

  • Validate linearity, sensitivity, and reproducibility

  • Calculate inter- and intra-assay coefficient of variation

• Mass spectrometry approaches:

  • Implement targeted proteomics using selected/multiple reaction monitoring

  • Use stable isotope-labeled peptide standards for accurate quantification

  • Focus on unique PTPN13 peptides verified by discovery proteomics

  • Apply appropriate normalization methods based on experimental design

• Comparison across techniques:

  • Validate results using orthogonal methods (WB, ELISA, MS)

  • Assess correlation between protein and mRNA expression

  • Consider post-translational modifications that may affect antibody recognition

How should researchers interpret discrepancies between PTPN13 expression data from different detection methods?

When faced with discrepancies in PTPN13 detection across methods:

• Technical considerations:

  • Epitope accessibility: Different antibodies (e.g., those targeting Met1~Asn161 vs. Met1-Arg500 ) may have variable access to epitopes depending on protein conformation

  • Protein size effects: The large size of PTPN13 (~260 kDa) may cause inefficient transfer in Western blots

  • Post-translational modifications: Phosphorylation or other modifications may mask epitopes in specific contexts

• Methodological analysis:

  • Compare detection limits across methods (Western blot vs. ELISA)

  • Assess linearity range for each quantification approach

  • Evaluate effects of sample preparation on epitope preservation

• Biological interpretation:

  • Consider isoform expression differences

  • Evaluate protein stability and turnover rates

  • Assess subcellular localization affecting extraction efficiency

• Resolution strategies:

  • Use multiple antibodies targeting different PTPN13 epitopes

  • Compare results from multiple sample preparation methods

  • Implement complementary detection technologies

  • Design validation experiments to resolve specific discrepancies

What approaches can researchers use to study PTPN13 interactions with multiple binding partners?

To investigate PTPN13's complex interactome:

• Co-immunoprecipitation strategies:

  • Use PTPN13 antibodies for pull-down experiments

  • Analyze precipitates by mass spectrometry to identify novel interactors

  • Confirm specific interactions with reverse co-IP experiments

  • Map interactions to specific PTPN13 domains (PDZ, phosphatase domain)

• Proximity-based methods:

  • Implement BioID or APEX2 proximity labeling with PTPN13 fusion proteins

  • Use FRET/BRET assays to monitor dynamic protein interactions

  • Apply proximity ligation assays for in situ visualization of protein complexes

• Domain-specific analysis:

  • Create domain deletion mutants to map interaction surfaces

  • Express individual PDZ domains to identify domain-specific binding partners

  • Analyze the effects of phosphatase activity on protein-protein interactions

• Functional validation:

  • Disrupt specific interactions using competitive peptides

  • Assess functional consequences of interrupted PTPN13 interactions

  • Correlate interaction profiles with cellular phenotypes (apoptosis resistance, migration)

• Computational approaches:

  • Use protein-protein interaction prediction algorithms

  • Apply molecular docking to model interaction interfaces

  • Integrate interaction data with functional genomics datasets