PTPN18 Antibody

What is PTPN18 and why is it important in research?

PTPN18, also known as BDP1 (Brain-Derived Phosphatase 1), is a non-receptor type protein tyrosine phosphatase belonging to the protein-tyrosine phosphatase family. The protein consists of 459 amino acids with no transmembrane domain and has a calculated molecular weight of approximately 50 kDa. PTPN18 contains a characteristic PEST motif and shares significant homology with members of the PTP-PEST gene family . This phosphatase has gained research significance due to its involvement in multiple signaling pathways and its emerging role in cancer biology, particularly colorectal cancer where it has been shown to promote development by stabilizing MYC protein levels and activating the MYC-CDK4 axis . Research into PTPN18 is crucial for understanding fundamental cellular signaling mechanisms and for identifying potential therapeutic targets in cancer treatment.

How do I select the appropriate PTPN18 antibody for my specific research application?

When selecting a PTPN18 antibody, consider these methodological factors:

• Application compatibility: Different antibodies perform optimally in specific applications. Based on validated data, ensure your selected antibody is tested for your intended application (WB, IHC, IF, or ELISA).

• Target epitope: Consider which region of PTPN18 you need to target. Some antibodies target specific amino acid regions (e.g., AA 1-351, AA 1-210, or C-terminal regions AA 430-459), which may affect recognition of specific isoforms or detection after post-translational modifications .

• Validated reactivity: Verify species cross-reactivity with your experimental model. Some PTPN18 antibodies are validated only for human samples, while others show reactivity with mouse or rat samples as well .

• Clonality consideration:

  • Polyclonal antibodies (like 17551-1-AP) offer broader epitope recognition

  • Monoclonal antibodies provide higher specificity for particular epitopes

• Published validation: Review literature where the antibody has been successfully used. For example, antibody 17551-1-AP has several publications documenting its use in WB, IHC, and IF applications .

What are the recommended dilutions for PTPN18 antibodies in different applications?

The optimal dilution of PTPN18 antibodies varies significantly depending on the specific application and sample type. For accurate results, it's essential to follow empirically determined dilution ranges and optimize for your specific experimental system.

For PTPN18 antibody 17551-1-AP, the following dilution ranges are recommended:

It is strongly recommended to perform a dilution series during initial optimization experiments to determine the optimal concentration for your specific sample type and experimental conditions . Sample-dependent variations are common, so checking validation data galleries for similar tissue or cell types can provide useful starting points. For example, this antibody has been positively tested in Western blots using Jurkat cells and human brain tissue .

How should I optimize immunohistochemistry protocols when using PTPN18 antibodies?

Optimizing immunohistochemistry protocols for PTPN18 detection requires careful attention to several key parameters:

• Antigen retrieval: For PTPN18 antibody 17551-1-AP, the suggested antigen retrieval method uses TE buffer at pH 9.0. Alternatively, citrate buffer at pH 6.0 can be used if the primary method doesn't yield satisfactory results . The choice between these methods may depend on tissue fixation conditions and embedding procedures.

• Antibody dilution: Start with the recommended range (1:20-1:200 for 17551-1-AP) and perform a dilution series to determine optimal concentration for your specific tissue. Higher concentrations may be needed for tissues with lower PTPN18 expression.

• Incubation conditions: For primary antibody, overnight incubation at 4°C often yields better results than shorter incubations at room temperature, especially for nuclear or low-abundance proteins like PTPN18.

• Detection system selection: For low-abundance targets, consider using amplification systems such as polymer-based detection or tyramide signal amplification.

• Positive control tissues: Human brain tissue has been validated for positive PTPN18 detection and should be considered as a positive control . Colorectal cancer tissues also show elevated PTPN18 expression as demonstrated in research studies .

• Protocol validation: Confirm specificity using appropriate controls, including:

  • Omission of primary antibody (negative control)

  • Tissue from PTPN18 knockout models if available

  • Comparison with other validated anti-PTPN18 antibodies

Optimization should be iterative, adjusting one parameter at a time while keeping others constant to isolate the effect of each change.

What experimental controls are essential when using PTPN18 antibodies?

When using PTPN18 antibodies, implementing proper controls is critical for ensuring data reliability and accurate interpretation:

• Positive controls: Include samples known to express PTPN18. For antibody 17551-1-AP, Jurkat cells and human brain tissue have been validated as positive control samples for Western blotting . For immunohistochemistry, human brain tissue is recommended as a positive control .

• Negative controls:

  • Primary antibody omission - perform parallel staining without the PTPN18 antibody to assess non-specific binding of the detection system

  • Isotype controls - use matched isotype (e.g., Rabbit IgG for 17551-1-AP) at the same concentration to evaluate non-specific binding

  • Tissue negative controls - include tissues known not to express PTPN18 or PTPN18-knockout tissue if available

• Loading/technical controls:

  • For Western blotting, include housekeeping protein controls like GAPDH, which was used in the colorectal cancer research studies

  • For immunohistochemistry, include parallel sections stained with established markers

  • For fluorescence applications, include appropriate autofluorescence controls

• Validation controls:

  • Peptide competition assay - pre-incubation of antibody with immunizing peptide should abolish specific staining

  • siRNA or CRISPR knockdown validation - compare staining in PTPN18-deficient cells, as demonstrated in the CRC research using CRISPR-Cas9 methodology

  • Multiple antibody validation - compare results with a second PTPN18 antibody targeting a different epitope

• Functional controls: In advanced research, include controls that demonstrate antibody functionality in specific contexts, such as immunoprecipitation followed by mass spectrometry to confirm the identity of the precipitated protein.

How can I troubleshoot weak or absent signals when using PTPN18 antibodies?

When encountering weak or absent signals with PTPN18 antibodies, employ this systematic troubleshooting approach:

• Sample preparation issues:

  • Protein degradation: Ensure use of fresh samples with protease inhibitors

  • Insufficient protein extraction: For PTPN18 (a 50 kDa protein), optimize lysis buffers for complete solubilization

  • Inadequate antigen retrieval: For IHC, test both recommended methods - TE buffer (pH 9.0) and citrate buffer (pH 6.0)

• Antibody-specific factors:

  • Dilution optimization: PTPN18 antibody 17551-1-AP should be titrated within the recommended ranges (WB: 1:500-1:3000; IHC: 1:20-1:200)

  • Incubation time extension: For low-abundance samples, increase primary antibody incubation (overnight at 4°C)

  • Antibody lot variation: Compare with previous lots or alternate antibodies like ABIN525721

• Detection system limitations:

  • Signal amplification: Implement more sensitive detection methods (enhanced chemiluminescence for WB, polymer-based detection for IHC)

  • Blocking optimization: Test alternative blocking reagents to reduce background while preserving specific signal

  • Secondary antibody concentration: Increase concentration if primary signal is confirmed present but weak

• Technical modifications:

  • For Western blot: Load more protein (50-100 μg), reduce washing stringency, extend exposure time

  • For IHC/IF: Increase antibody concentration, extend DAB development, use tyramide signal amplification

  • For all applications: Reduce detergent concentration in washing buffers

• Control experiments:

  • Run positive control samples (Jurkat cells or human brain tissue for 17551-1-AP)

  • Verify PTPN18 expression levels in your sample type using qPCR before protein detection

  • Consider that PTPN18 expression varies significantly between tissues and may be upregulated in conditions like colorectal cancer

If these approaches fail, consider testing alternative PTPN18 antibodies that target different epitopes or employing tagged overexpression systems to verify detection capability.

What experimental approaches are most effective for studying PTPN18's function in cancer cells?

To effectively investigate PTPN18's function in cancer cells, researchers should employ a comprehensive experimental strategy:

This multi-faceted approach provides both mechanistic insights and clinical relevance for understanding PTPN18's role in cancer biology.

How can substrate-trapping mutants be used to identify PTPN18 targets?

Substrate-trapping mutants represent a sophisticated approach for identifying physiological substrates of protein tyrosine phosphatases like PTPN18. The methodology involves several critical considerations:

• Mutant design principles:
  The PTPN18 D90A C122S double mutant exemplifies effective substrate-trapping design by combining two critical mutations :

  • WPD loop mutation (D90A): The aspartate in the "WPD loop" is mutated to alanine, eliminating the catalytic acid required for efficient phosphate release

  • Signature motif mutation (C122S): The catalytic cysteine in the signature motif is replaced with serine, preventing formation of the phospho-enzyme intermediate

  This combination creates a "substrate-trapping" mutant that binds phosphorylated substrates with high affinity but cannot complete the dephosphorylation cycle, effectively capturing substrates.

• Experimental setup for substrate identification:

  • Expression system: Generate cell lines expressing FLAG-tagged wild-type PTPN18 and FLAG-tagged PTPN18 D90A C122S using lentiviral transfection

  • Functional validation: Confirm the mutant's loss of phosphatase activity using in vitro phosphatase assays

  • Stimulation conditions: Treat cells with appropriate stimuli to increase tyrosine phosphorylation (e.g., pervanadate, growth factors)

• Substrate capture and identification:

  • Immunoprecipitation: Use anti-FLAG antibodies to pull down PTPN18 complexes

  • Comparative analysis: Compare proteins co-immunoprecipitating with the substrate-trapping mutant versus wild-type PTPN18

  • Western blotting: Probe for suspected substrates or use anti-phosphotyrosine antibodies

  • Mass spectrometry: For unbiased identification of captured phosphoproteins

• Validation of potential substrates:

  • Reciprocal co-immunoprecipitation: Confirm interaction by immunoprecipitating the candidate substrate

  • Direct dephosphorylation assays: Test if wild-type PTPN18 can dephosphorylate the candidate in vitro

  • Cellular validation: Examine phosphorylation status of candidates in PTPN18 knockout versus wildtype cells

  • Functional studies: Assess whether phosphorylation-defective mutants of the substrate mimic PTPN18 effects

• Application to cancer research:
  The colorectal cancer study used this approach to investigate PTPN18's interactions with the MYC-CDK4 axis, demonstrating that PTPN18 stabilizes MYC protein levels . This substrate-trapping strategy revealed that while PTPN18's phosphatase activity is important for its function, the D90A C122S mutant could still form protein-protein interactions, helping distinguish between catalytic and scaffolding roles.

This methodology represents a powerful approach for mapping the substrate landscape of PTPN18 and understanding its mechanistic contributions to both normal cellular processes and disease states.

What is known about PTPN18's role in colorectal cancer progression?

PTPN18 has emerged as a significant contributor to colorectal cancer (CRC) development and progression through several key mechanisms:

These findings collectively establish PTPN18 as a novel oncogenic factor in colorectal cancer, positioning it as a potential therapeutic target for future CRC treatment strategies.

How does the MYC-CDK4 axis mediate PTPN18's effects in cancer cells?

The MYC-CDK4 axis represents a critical mechanism through which PTPN18 exerts its oncogenic effects in colorectal cancer cells:

• PTPN18-MYC interaction and stabilization:

  • Direct interaction: Research has demonstrated that PTPN18 physically interacts with MYC protein in colorectal cancer cells

  • Protein stabilization: PTPN18 significantly increases MYC protein levels without affecting mRNA expression, indicating post-translational regulation

  • Mechanistic basis: While the exact mechanism remains under investigation, PTPN18 likely prevents MYC degradation either by direct dephosphorylation of destabilizing phosphorylation sites or by interfering with ubiquitin-proteasome targeting

• CDK4 upregulation as a downstream consequence:

  • Transcriptional activation: Stabilized MYC, a potent transcription factor, enhances CDK4 expression

  • Expression correlation: PTPN18 knockout in HCT116 cells leads to decreased MYC levels with concomitant reduction in CDK4 expression

  • In vivo validation: Xenograft tumors derived from PTPN18-knockout cells showed significantly reduced levels of both MYC and CDK4 proteins

• Cell cycle impact and proliferation effects:

  • CDK4 function: As a critical regulator of G1/S transition, CDK4 upregulation accelerates cell cycle progression

  • Proliferative outcome: The enhanced MYC-CDK4 signaling drives increased cancer cell proliferation in both in vitro and in vivo models

  • Molecular signaling cascade: PTPN18 → MYC stabilization → CDK4 upregulation → accelerated cell cycle → enhanced proliferation

• Dependency on phosphatase activity:

  • Catalytic requirement: The phosphatase-dead PTPN18 D90A C122S mutant fails to promote proliferation, suggesting that PTPN18's enzymatic activity is essential for MYC stabilization

  • Substrate specificity: This indicates that specific dephosphorylation events, rather than mere protein binding, are required for PTPN18's oncogenic function

• Therapeutic implications:

  • Targeting vulnerability: The PTPN18-MYC-CDK4 axis represents a potential target for therapeutic intervention

  • Dual approach potential: Strategies could target either PTPN18 activity directly or the downstream MYC-CDK4 pathway

  • Biomarker utility: PTPN18 expression levels could serve as a biomarker for identifying tumors likely to respond to CDK4 inhibitors

This mechanistic understanding provides deeper insight into how a single phosphatase can orchestrate complex oncogenic signaling networks and identifies multiple points for potential therapeutic intervention in colorectal cancer.

How should researchers interpret varying PTPN18 expression levels in different tissues?

When interpreting varying PTPN18 expression levels across different tissues and contexts, researchers should consider several critical factors:

What are common pitfalls in interpreting PTPN18 antibody-based experimental results?

Researchers should be aware of several common pitfalls when interpreting PTPN18 antibody-based experimental results:

To minimize these pitfalls, researchers should implement rigorous controls, validate findings using complementary techniques, consider both expression and activity measurements, and interpret results within the appropriate biological context.

How can researchers effectively design knockdown/knockout experiments to study PTPN18 function?

Designing effective knockdown/knockout experiments for PTPN18 functional studies requires meticulous planning and execution:

• Strategy selection based on experimental goals:

  • CRISPR-Cas9 knockout: For complete gene elimination when studying essential functions. In colorectal cancer research, this approach successfully demonstrated PTPN18's role in promoting tumor growth

  • siRNA/shRNA knockdown: For temporal studies or when complete knockout is lethal

  • Inducible systems: For studying developmental or stage-specific effects

  • Domain-specific mutations: For separating catalytic versus scaffolding functions, as demonstrated with the phosphatase-dead PTPN18 D90A C122S mutant

• CRISPR-Cas9 knockout design considerations:

  • Guide RNA (gRNA) design:

    • Target early exons to ensure complete protein disruption

    • Design multiple gRNAs to increase success probability

    • Verify minimal off-target effects using prediction tools

  • Validation requirements:

    • Confirm editing by genomic sequencing

    • Verify complete protein loss by Western blot

    • Establish multiple independent knockout clones to control for clonal variations

• RNAi knockdown optimization:

  • siRNA design:

    • Target conserved regions

    • Test multiple sequences for optimal knockdown

    • Use validated sequences when available

  • Delivery optimization:

    • Adjust transfection conditions for each cell type

    • Determine optimal concentration balancing efficiency and toxicity

    • Establish knockdown timeline through time-course experiments

  • Controls implementation:

    • Include non-targeting siRNA controls

    • Perform rescue experiments with siRNA-resistant constructs

    • Quantify knockdown efficiency by qPCR and Western blot

• Functional readout selection:

  • Proliferation assays: CCK8, colony formation, and soft agar assays effectively demonstrated PTPN18's growth-promoting effects

  • Signaling pathway analysis: Western blotting for MYC and CDK4 revealed the mechanism of PTPN18 action

  • Phenotypic assays: Select readouts relevant to hypothesized function (e.g., migration, invasion, apoptosis)

  • In vivo validation: Xenograft models provided critical confirmation of in vitro findings in CRC research

• Mechanistic investigation design:

  • Substrate identification: Employ substrate-trapping mutants (PTPN18 D90A C122S) to capture physiological substrates

  • Protein interaction studies: Co-immunoprecipitation to identify binding partners like MYC

  • Rescue experiments: Reintroduce wild-type or mutant PTPN18 to determine domain-specific contributions

  • Epistasis analysis: Manipulate downstream effectors (e.g., MYC or CDK4) in PTPN18-deficient backgrounds to establish pathway hierarchies

By systematically implementing these design principles, researchers can generate robust and reproducible data on PTPN18 function while avoiding common pitfalls in gene manipulation studies.