PTPRK Antibody

What is PTPRK and why is it significant for cell biology research?

PTPRK (Protein Tyrosine Phosphatase Receptor Type K) is a receptor tyrosine phosphatase that plays critical roles in regulating cell-cell adhesion, growth factor signaling, and tumor suppression. PTPRK is a transmembrane protein that becomes stabilized at cell-cell contacts through trans homophilic interactions. It functions as a negative regulator of EGFR signaling and forms complexes with β-catenin and γ-catenin/plakoglobin . Structurally, PTPRK exists as a 210 kDa precursor protein that undergoes processing by furin to form a mature heterodimeric protein consisting of a non-covalently attached amino-terminal extracellular subunit (E-subunit, 120 kDa) and a carboxyl-terminal transmembrane subunit (P-subunit, 95 kDa) . Recent research has demonstrated its importance in epithelial tissue integrity and its potential role as a tumor suppressor in multiple cancer types, making it a significant target for investigating disease mechanisms .

What are the essential considerations when selecting a PTPRK antibody for research?

When selecting a PTPRK antibody, researchers should consider:

The selection should be guided by the specific experimental question, tissue/cell type, and methodological approach. Consider antibodies that have been validated in multiple applications if your research involves diverse techniques .

How can I validate PTPRK antibody specificity in my experimental system?

Validating PTPRK antibody specificity requires multiple complementary approaches:

• CRISPR/Cas9 knockout controls: Generate PTPRK knockout cell lines to serve as negative controls, as demonstrated in studies using MCF10A epithelial cells . The complete absence of signal in knockout cells provides strong evidence of specificity.

• Colocalization studies: Examine whether PTPRK staining patterns align with known localization at cell-cell contacts. Using structured illumination microscopy, PTPRK has been shown to localize to puncta at basal cell-cell contacts that partially overlap with E-cadherin .

• Density-dependent expression: PTPRK protein levels increase with cell density, so comparing expression in sparse versus confluent cultures can serve as an internal validation .

• Western blot molecular weight verification: Verify that the detected protein corresponds to the expected molecular weight range (100-120 kDa for processed forms) .

• Co-culture experiments: Mix wildtype and PTPRK-knockout cells (labeled for identification) and confirm antibody staining is absent at contacts between wildtype and knockout cells .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide prior to application to verify that the signal is specifically blocked.

What are the recommended dilutions and sample preparation methods for PTPRK detection?

Optimal dilutions and sample preparation methods vary by application:

Western Blot:

• Recommended dilution: 1:500-1:1000

• Sample preparation:

  • Lyse cells in RIPA buffer supplemented with protease and phosphatase inhibitors

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

  • Load 20-50 μg of total protein per lane

  • Transfer to PVDF membrane (preferred over nitrocellulose for PTPRK)

Immunohistochemistry (IHC-P):

• Recommended dilution: 1:500

• Sample preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Perform heat-mediated antigen retrieval with citrate buffer (pH 6.0)

  • Block endogenous peroxidase and non-specific binding

Immunofluorescence/ICC:

• Recommended dilution: 2 μg/ml

• Sample preparation:

  • Fix cells with 4% paraformaldehyde for 15 minutes

  • Permeabilize with 0.1% Triton X-100

  • Block with 1-5% BSA or normal serum

Optimal dilutions should be determined empirically for each new lot of antibody and experimental system .

How can I use PTPRK antibodies to investigate its role in tumor suppression?

PTPRK has been implicated as a tumor suppressor in several cancer types, particularly colorectal cancer . To investigate this role:

• Expression correlation studies: Use PTPRK antibodies in tissue microarray analysis to correlate expression levels with patient survival outcomes. Loss of PTPRK activity has been observed in pancreatic cancer, primary CNS lymphoma, and melanoma, and is associated with poor survival .

• Substrate identification: Combine PTPRK antibodies with phosphoproteomic approaches to identify and validate substrate proteins. Studies have shown that PTPRK selectively dephosphorylates substrates including Afadin, PARD3, and δ-catenin family members, all important cell-cell adhesion regulators .

• 3D culture models: Use PTPRK antibodies in immunofluorescence studies of 3D spheroid cultures to assess morphological changes. Loss of PTPRK phosphatase activity leads to disrupted cell junctions and increased invasive characteristics, which can be visualized in these models .

• EMT marker correlation: Analyze the relationship between PTPRK expression and epithelial-to-mesenchymal transition markers. PTPRK knockout cells show downregulation of epithelial markers like Keratin14 and upregulation of mesenchymal markers like vitronectin (VTN) .

• TGFβ signaling interaction: As PTPRK is a TGFβ target gene, investigate its role in negative feedback regulation of TGFβ-induced EMT using antibodies to monitor expression changes in response to TGFβ treatment .

What approaches can I use to distinguish between catalytic and non-catalytic functions of PTPRK?

Recent research has revealed that PTPRK has both catalytic (phosphatase-dependent) and non-catalytic functions . To differentiate between these:

• Phosphatase-dead mutants: Generate cell lines expressing catalytically inactive PTPRK (typically by mutating the catalytic cysteine residue) and compare with wild-type PTPRK expression using antibodies to detect differential effects.

• Domain-specific antibodies: Use antibodies targeting different domains of PTPRK to investigate domain-specific functions. Antibodies recognizing the extracellular domain can assess adhesion functions, while those targeting the phosphatase domain can be used in activity assays .

• Substrate trapping: Employ substrate-trapping mutants of PTPRK (typically D→A mutations in the catalytic domain) combined with immunoprecipitation and phosphotyrosine immunoblotting to identify direct substrates.

• Proximity labeling: Use BioID or APEX2 fusion proteins with PTPRK to identify proteins in proximity that may be involved in non-catalytic scaffolding functions, followed by antibody validation of interactions.

• Comparative phosphoproteomics: Compare phosphoproteomic profiles of cells expressing wild-type PTPRK, phosphatase-dead PTPRK, and PTPRK knockout to differentiate between catalytic and non-catalytic effects on the phosphoproteome.

How can I investigate the role of PTPRK in cell-cell adhesion and collective cell migration?

PTPRK regulates cell-cell adhesion and promotes collective, directed migration in colorectal cancer cells . To investigate these functions:

• Live-cell imaging with tagged antibodies: Use fluorescently labeled PTPRK antibody fragments (Fab) to track PTPRK dynamics during cell migration without interfering with function.

• Wound healing assays: Perform scratch assays on control and PTPRK-depleted cells, then use antibodies to examine localization of PTPRK and adhesion components during wound closure. Studies have shown that PTPRK knockout cells exhibit impaired wound-healing responses .

• FRAP (Fluorescence Recovery After Photobleaching): After immunolabeling, use FRAP to measure PTPRK mobility at cell-cell contacts, providing insights into its stabilization through homophilic interactions.

• Co-immunoprecipitation: Use PTPRK antibodies for co-IP experiments to identify adhesion complex components that interact with PTPRK under different conditions (e.g., calcium depletion, growth factor stimulation).

• Transepithelial electrical resistance (TEER): Measure epithelial barrier integrity in control versus PTPRK-depleted cells, then use antibodies to correlate barrier function with PTPRK localization and substrate phosphorylation status.

• 3D invasion assays: Employ PTPRK antibodies in immunofluorescence to visualize its distribution in invading versus non-invading regions of 3D cultures, as loss of PTPRK has been linked to increased invasive characteristics .

What methods can I use to study PTPRK homophilic interactions?

PTPRK is stabilized at the plasma membrane by trans homophilic interactions upon cell-cell contact . To study these interactions:

• Mixed culture experiments: Co-culture wildtype cells with PTPRK knockout cells (labeled with nuclear markers for identification) and use immunostaining to demonstrate that PTPRK is absent from cell-cell contacts between wildtype and adjacent knockout cells .

• Density-dependent expression analysis: Culture cells at different densities and use western blotting to demonstrate that PTPRK protein levels increase with increasing cell density .

• FRET (Förster Resonance Energy Transfer): Label PTPRK with donor and acceptor fluorophores to detect homophilic interactions between adjacent cells using FRET microscopy.

• Recombinant protein interaction assays: Express the PTPRK extracellular domain as a recombinant protein and use protein microarrays to screen for potential ligands .

• Bead aggregation assays: Couple recombinant PTPRK extracellular domain to fluorescent beads and assess homophilic aggregation, similar to techniques used for other receptor protein tyrosine phosphatases in the R2B family .

• Super-resolution microscopy: Utilize structured illumination microscopy to visualize PTPRK localization at cell-cell contacts with high precision, as demonstrated in MCF10A epithelial cells where PTPRK localizes to puncta at basal cell-cell contacts .

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

Optimizing IHC protocols for PTPRK detection requires attention to several critical factors:

• Antigen retrieval: Heat-mediated antigen retrieval with citrate buffer (pH 6.0) is recommended. Perform retrieval before commencing with the IHC staining protocol .

• Antibody dilution: Start with a 1:500 dilution for paraffin-embedded sections and adjust as needed based on signal intensity and background .

• Incubation conditions: Overnight incubation at 4°C often yields better results than shorter incubations at room temperature.

• Detection system: HRP-based detection systems with tyramide signal amplification can enhance sensitivity for detecting lower expression levels.

• Controls: Include positive control tissues known to express PTPRK (e.g., spleen tissue) and negative controls (primary antibody omission and ideally PTPRK knockout tissues).

• Signal enhancement: For weakly expressed PTPRK, consider using polymer-based detection systems or biotin-streptavidin amplification, being mindful of potential background issues.

• Counterstaining: Light hematoxylin counterstaining helps visualize tissue architecture without obscuring specific PTPRK staining.

• Multiplex staining: When co-staining with other markers, use antibodies raised in different host species to avoid cross-reactivity and employ appropriate sequential staining protocols.

What are the best approaches for troubleshooting Western blot issues with PTPRK antibodies?

Common Western blot issues with PTPRK antibodies and their solutions include:

For PTPRK specifically, recommended dilutions for Western blot are 1:500-1:1000 . Testing antibodies against mouse or rat spleen tissue can serve as positive controls .

How can I optimize immunofluorescence protocols to visualize PTPRK at cell-cell junctions?

To achieve optimal visualization of PTPRK at cell-cell junctions:

• Cell culture conditions: Grow cells to full confluence (90-100%) to ensure formation of mature cell-cell contacts, as PTPRK is stabilized at these locations .

• Fixation method: Use 4% paraformaldehyde for 15 minutes at room temperature; avoid methanol fixation which can disrupt membrane protein epitopes.

• Permeabilization: Gentle permeabilization with 0.1% Triton X-100 for 5 minutes preserves junction structures better than stronger detergents.

• Antibody selection: Use antibodies targeting the extracellular domain (e.g., AA 589-749) for better access to junction-localized PTPRK .

• Antibody concentration: A concentration of 2 μg/ml has been successfully used for immunofluorescence applications .

• Blocking: Extensive blocking (1 hour, room temperature) with 5% normal serum from the secondary antibody host species reduces background.

• Advanced microscopy: Structured illumination microscopy has successfully visualized PTPRK localization to puncta at basal cell-cell contacts that partially overlap with E-cadherin in epithelial cells .

• Co-staining controls: Include co-staining with established junction markers like E-cadherin to confirm proper junction visualization .

• Z-stack imaging: Collect Z-stack images to fully capture PTPRK distribution throughout the cell-cell contact areas, as junctional localization can vary with depth.

What controls should I include when using PTPRK antibodies in experimental workflows?

Comprehensive controls for PTPRK antibody experiments include:

• Genetic controls:

  • PTPRK knockout cells or tissues as negative controls

  • PTPRK overexpression systems as positive controls

  • Rescue experiments with re-expression of PTPRK in knockout backgrounds

• Antibody controls:

  • Primary antibody omission to assess secondary antibody non-specific binding

  • Isotype controls (rabbit IgG) at the same concentration as the primary antibody

  • Peptide competition assays using the immunizing peptide (when available)

• Technical controls:

  • Multiple antibodies targeting different PTPRK regions to confirm concordant results

  • Correlation of protein detection with mRNA expression (RT-qPCR)

  • Cross-validation with multiple techniques (e.g., IF, WB, IHC)

• Biological controls:

  • Density-dependent expression analysis (PTPRK increases with cell density)

  • Co-culture experiments with wildtype and knockout cells to demonstrate homophilic binding specificity

  • Positive control tissues known to express PTPRK (e.g., spleen tissue)

• Application-specific controls:

  • For phosphatase activity studies: catalytically inactive PTPRK mutants

  • For substrate identification: substrate-trapping mutants

  • For homophilic interaction studies: cells expressing only the extracellular domain

How are PTPRK antibodies being used to study its role in colorectal cancer?

Recent research has employed PTPRK antibodies to investigate its tumor suppressor function in colorectal cancer through several approaches:

• Expression profiling: PTPRK antibodies are used in tissue microarrays to correlate expression levels with clinical outcomes. PTPRK has been implicated as a tumor suppressor in colorectal cancer, and mutations and genetic events inactivating PTPRK are found in human colorectal cancers .

• Functional studies: Antibodies help visualize cellular changes in PTPRK knockout or phosphatase-dead mutant colorectal cancer cell lines, revealing that loss of PTPRK phosphatase activity leads to disrupted cell junctions and increased invasive characteristics .

• RSPO3 fusion identification: PTPRK antibodies aid in detecting specific gene fusions where the PTPRK promoter drives the expression of oncogenic RSPO3 in a subset of colorectal cancers .

• Cell migration and invasion: Antibodies are utilized to study how PTPRK regulates cell adhesion signaling, suppresses invasion, and promotes collective, directed migration in colorectal cancer cells .

• EMT marker correlation: PTPRK knockout cells show downregulation of epithelial markers and upregulation of mesenchymal markers, suggesting PTPRK suppresses epithelial-to-mesenchymal transition (EMT). Antibodies help validate these expression changes in vitro and in patient samples .

• TGFβ signaling interaction: As PTPRK is a TGFβ target gene, antibodies help investigate its role in negative feedback of TGFβ-induced EMT, which could have pathological effects if perturbed .

What are the cutting-edge methods for studying PTPRK substrates and signaling mechanisms?

Advanced methodologies for investigating PTPRK substrates and signaling pathways include:

• Quantitative phosphoproteomics: Combining SILAC (Stable Isotope Labeling with Amino acids in Cell culture) with phosphotyrosine enrichment to identify proteins with altered phosphorylation status in PTPRK knockout versus wildtype cells .

• Proximity labeling: BioID or APEX2 tagging of PTPRK to identify proximal proteins in the cellular environment, which has helped identify high confidence PTPRK substrates .

• Direct dephosphorylation assays: In vitro assays using recombinant PTPRK phosphatase domain and phosphorylated substrates to confirm direct dephosphorylation, which has validated several substrates including Afadin, PARD3, and δ-catenin family members .

• Substrate trapping: Using substrate-trapping mutants of PTPRK to capture physiological substrates that would normally be dephosphorylated and released.

• CRISPR/Cas9 gene editing: Generation of endogenously tagged PTPRK to study native protein interactions without overexpression artifacts.

• Phospho-specific antibodies: Development of antibodies specifically recognizing phosphorylated forms of PTPRK substrates to monitor PTPRK activity in situ.

• Interactome mapping: Combining immunoprecipitation with mass spectrometry to comprehensively identify PTPRK-interacting proteins under different cellular conditions.

• Live-cell FRET sensors: Genetically encoded FRET sensors to monitor PTPRK-dependent dephosphorylation events in real-time in living cells.

How can I design experiments to investigate PTPRK's role in inflammatory bowel disease and celiac disease?

PTPRK has been linked to inflammatory bowel disease susceptibility and celiac disease through genetic studies . To investigate these connections:

• SNP functional analysis: Use PTPRK antibodies to compare expression levels and localization patterns between cells carrying disease-associated versus non-associated SNPs.

• Intestinal organoid models: Apply PTPRK antibodies in immunofluorescence studies of intestinal organoids derived from patients with and without disease to examine PTPRK expression and localization patterns.

• Barrier function assays: Measure transepithelial electrical resistance (TEER) in control versus PTPRK-depleted intestinal epithelial cell monolayers, correlating PTPRK expression (detected by antibodies) with barrier integrity.

• Inflammatory mediator response: Use antibodies to track PTPRK expression and localization changes in response to inflammatory cytokines relevant to IBD and celiac disease.

• PTPRK substrate phosphorylation: Employ phospho-specific antibodies against PTPRK substrates to examine their phosphorylation status in intestinal tissues from patients versus controls.

• Gluten response studies: In celiac disease models, examine PTPRK expression and localization changes in response to gliadin peptides, the toxic components of gluten.

• Immune cell interaction models: Co-culture intestinal epithelial cells with immune cells and use PTPRK antibodies to investigate its role in epithelial-immune cell communication during inflammation.

• Genetic modification in mouse models: Generate intestine-specific PTPRK knockout or disease-associated SNP knock-in mice and use antibodies to validate the models and study disease pathology.

What are the emerging applications of PTPRK antibodies in tissue repair research?

Recent research has highlighted PTPRK's role in tissue repair, opening new avenues for antibody applications:

• Wound healing models: PTPRK antibodies help visualize its distribution during wound closure, as knockout cells show impaired wound-healing responses likely due to effects on coordinated cell movement and cellular polarization .

• Epithelial barrier recovery: Antibodies track PTPRK expression and localization during epithelial barrier restoration after injury, which is relevant to inflammatory bowel disease research .

• Cell reprogramming studies: PTPRK is involved in regulating epithelial-mesenchymal transition (EMT), so antibodies help monitor its expression during cell state transitions relevant to repair processes .

• Organoid development: Antibodies visualize PTPRK distribution during organoid formation and maturation, providing insights into its role in tissue architecture establishment.

• In vivo injury models: PTPRK antibodies are used in immunohistochemical analysis of tissue sections from injury models (e.g., colitis models) to track expression changes during the repair process.

• Mechanotransduction research: As cell-cell adhesion is mechanosensitive, antibodies help investigate how PTPRK localization and activity respond to mechanical forces during tissue repair.

• Therapeutic targeting validation: Antibodies validate the specificity of approaches targeting PTPRK to enhance tissue repair, potentially through promoting cell junction stability and collective migration .

• Cancer treatment response: PTPRK antibodies help monitor expression changes during tissue recovery after cancer treatments, which may inform strategies to reduce treatment-associated tissue damage.