PTPRB Antibody

What is PTPRB and why are PTPRB antibodies important in research?

PTPRB, or Protein tyrosine Phosphatase Receptor Type B, is a receptor-type protein tyrosine phosphatase involved in critical cellular processes. This protein plays significant roles in vascular development and has been implicated in cancer progression. PTPRB functions by dephosphorylating various receptor tyrosine kinases including TEK (Tie2), VE-Cadherin, VEGFR2, and FGD5 . The importance of PTPRB antibodies stems from their ability to enable researchers to detect, localize, and study the expression patterns of this phosphatase in various tissues and experimental conditions.

Recent research has demonstrated that PTPRB promotes metastasis of colorectal cancer (CRC) via inducing epithelial-mesenchymal transition (EMT), marking it as a potential therapeutic target . Additionally, studies show that targeting the vascular-specific phosphatase PTPRB can protect against developmental defects in mice models with reduced TEK signaling . These findings highlight the significance of PTPRB antibodies as tools for understanding disease mechanisms and developing potential therapeutic approaches.

What are the key specifications to consider when selecting a PTPRB antibody?

When selecting a PTPRB antibody for research applications, several critical specifications require consideration to ensure experimental success. First, researchers should identify the target species reactivity needed for their experimental model. Available PTPRB antibodies demonstrate reactivity against human, mouse, and/or rat PTPRB, with some antibodies showing cross-reactivity between species .

Second, the binding specificity region matters significantly. Different antibodies target distinct amino acid regions of PTPRB, such as AA 1655-1918, AA 280-360, AA 1041-1310, or N-terminal regions . The selection of the target region should align with the specific research questions and experimental constraints, particularly if certain domains of PTPRB are of special interest.

Third, researchers must consider antibody clonality. Most available PTPRB antibodies are polyclonal, typically raised in rabbit hosts, although goat-derived antibodies are also available . The choice between polyclonal and monoclonal antibodies depends on the experimental requirements for specificity versus sensitivity. Finally, researchers should evaluate the validated applications for each antibody, as different PTPRB antibodies are optimized for specific techniques including Western Blotting (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP), Immunocytochemistry (ICC), and ELISA .

How does PTPRB expression differ between normal and pathological tissues?

PTPRB expression exhibits significant differences between normal and pathological tissues, particularly in cancer contexts. Research has demonstrated that PTPRB is expressed at significantly higher levels in colorectal cancer (CRC) tissues compared to adjacent non-tumor tissues . This upregulation appears to correlate with the invasive potential of cancer cells, as PTPRB is also expressed at higher levels in CRC cell lines with enhanced invasive capabilities .

The pathological significance of increased PTPRB expression relates to its role in promoting epithelial-mesenchymal transition (EMT), a critical process in cancer metastasis. Studies show that PTPRB overexpression decreases vimentin expression and promotes E-cadherin expression, molecular changes consistent with EMT progression . Furthermore, the relationship between PTPRB expression and hypoxic conditions provides insight into tumor microenvironment dynamics. Hypoxia can induce EMT and promote invasion in CRC cells, but interestingly, these effects can be eliminated by PTPRB knockdown . This suggests that PTPRB may function as a mediator of hypoxia-induced metastatic behavior in colorectal cancer, highlighting its significance in pathological processes and its potential as a therapeutic target.

What are the optimal protocols for using PTPRB antibodies in Western blotting?

Western blotting with PTPRB antibodies requires careful optimization to achieve reliable and reproducible results. Based on manufacturer recommendations, the optimal dilution range for PTPRB antibodies in Western blotting typically falls between 1:500-2000 . This range provides a starting point, but researchers should conduct titration experiments to determine the ideal concentration for their specific experimental conditions and antibody lot.

When performing Western blotting for PTPRB, it's important to note the expected molecular weight of the target protein. The observed molecular weight of PTPRB in Western blots is approximately 111 kDa, although the calculated molecular weight is significantly higher at 224,301 Da . This discrepancy likely results from post-translational modifications, alternative splicing, or proteolytic processing. Researchers should include appropriate positive controls, such as cell lines known to express PTPRB, to validate band specificity.

For sample preparation, standard protein extraction methods using RIPA buffer supplemented with protease and phosphatase inhibitors are generally suitable. Given that PTPRB is a membrane-associated receptor-type phosphatase, particular attention should be paid to membrane protein extraction efficiency. Additionally, optimization of transfer conditions is crucial for large proteins like PTPRB, with extended transfer times or specialized transfer buffers often yielding better results for high molecular weight proteins.

How can researchers effectively employ PTPRB antibodies in immunohistochemistry studies?

Immunohistochemistry (IHC) using PTPRB antibodies requires specific methodological considerations to achieve optimal staining and reliable results. Most commercially available PTPRB antibodies have been validated for IHC applications, with both paraffin-embedded and frozen tissue sections yielding satisfactory results . For paraffin-embedded tissues, researchers should optimize antigen retrieval methods, typically employing either heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0).

Blocking steps are crucial for reducing non-specific binding, particularly when using polyclonal antibodies. A blocking solution containing 5-10% normal serum (from the same species as the secondary antibody) and 1% BSA in PBS is generally effective. The optimal PTPRB antibody dilution for IHC applications typically ranges from 1:100 to 1:500, but this should be determined empirically for each specific antibody and tissue type .

For visualization, both chromogenic and fluorescent detection methods are compatible with PTPRB antibodies. When interpreting results, researchers should be aware of the expected subcellular localization of PTPRB, which is primarily membrane-associated with potential cytoplasmic components. Appropriate positive controls (such as vascular endothelial cells or colorectal cancer tissue sections) and negative controls (including isotype controls and antibody pre-absorption with immunizing peptide) should be included in all experiments to validate staining specificity and optimize protocol conditions.

What are the key considerations for measuring PTPRB phosphatase activity in experimental models?

Measuring PTPRB phosphatase activity presents unique challenges due to the complexity of phosphatase biochemistry and the specificity of substrate interactions. While direct phosphatase activity assays are not detailed in the search results, researchers can employ indirect approaches to assess PTPRB function. One effective strategy involves measuring the phosphorylation status of known PTPRB substrates, particularly TEK (Tie2). In vivo studies have utilized immunoprecipitation assays to quantify TEK phosphorylation levels in lung tissue, demonstrating that Ptprb heterozygosity (approximately 50% reduction in PTPRB expression) results in a 118% increase in phosphorylated TEK .

For cellular models, researchers can implement knockdown or overexpression approaches to modulate PTPRB levels, followed by assessment of substrate phosphorylation. Techniques such as phospho-specific Western blotting, phospho-flow cytometry, or phospho-specific ELISA can effectively quantify changes in substrate phosphorylation. When designing such experiments, careful consideration of experimental controls is essential. These should include wild-type cells/tissues, phosphatase-dead PTPRB mutants (for overexpression studies), and non-targeting controls (for knockdown approaches).

Additionally, researchers investigating PTPRB function should consider the potential impact of experimental conditions on phosphatase activity. Factors such as cell confluence, growth factor stimulation, hypoxia, and other microenvironmental conditions may significantly influence PTPRB activity and its biological consequences. For instance, hypoxic conditions have been shown to induce EMT and promote invasion in CRC cells, effects that are eliminated by PTPRB knockdown , suggesting that oxygen tension may affect PTPRB function.

How can PTPRB antibodies be utilized to investigate cancer metastasis mechanisms?

PTPRB antibodies serve as powerful tools for investigating the molecular mechanisms underlying cancer metastasis, particularly in colorectal cancer research. Studies have established that PTPRB promotes metastasis of colorectal cancer cells by inducing epithelial-mesenchymal transition (EMT), a critical process in cancer progression . Researchers can employ PTPRB antibodies in multiple experimental approaches to elucidate these mechanisms. Immunohistochemical analysis using PTPRB antibodies allows researchers to compare expression levels between primary tumors, metastatic lesions, and adjacent normal tissues, providing insight into the correlation between PTPRB expression and metastatic potential .

For mechanistic studies, combining PTPRB antibodies with antibodies against EMT markers (E-cadherin, vimentin, TWIST1) in co-immunoprecipitation experiments can reveal protein-protein interactions involved in metastasis signaling pathways. Research has demonstrated that PTPRB overexpression decreases vimentin expression and promotes E-cadherin expression, molecular changes consistent with EMT progression . Furthermore, EMT blockade via TWIST1 knockdown inhibits migration and invasiveness of CRC cells, and even increased PTPRB expression cannot reverse this effect, suggesting a complex relationship between PTPRB and TWIST1 in the EMT pathway .

In functional studies, researchers can manipulate PTPRB expression through overexpression or knockdown approaches and utilize antibodies to validate the expression changes before assessing effects on invasion and metastasis in both in vitro and in vivo models. Such experimental designs have revealed that PTPRB knockdown decreases the number of invasive CRC cells in wound healing models and reduces tumor metastasis in vivo, while overexpression promotes invasion and metastasis .

What role does PTPRB play in vascular development and how can antibodies help elucidate this function?

PTPRB plays a critical role in vascular development through its regulation of tyrosine phosphorylation in key signaling pathways. PTPRB antibodies enable researchers to investigate these functions through various experimental approaches. Immunohistochemical and immunofluorescence studies utilizing PTPRB antibodies can visualize the spatiotemporal expression patterns of this phosphatase during different stages of vascular development, providing insight into its regulatory function in angiogenesis and vessel maturation .

Research has demonstrated that PTPRB acts on multiple vascular signaling components, including TEK (Tie2), VE-Cadherin, VEGFR2, and FGD5 . Of particular significance is its role in regulating TEK phosphorylation, which has direct implications for vascular development. Studies in mouse models have shown that deletion of a single allele of the Ptprb gene results in approximately 50% reduction in PTPRB protein expression and a corresponding 118% increase in phosphorylated TEK in lung tissue . This alteration in phosphorylation status has functional consequences for vascular structures, as evidenced by studies of Schlemm's canal (SC) development in the eye.

In a mouse model with Tek haploinsufficiency (Tek +/-), which normally exhibits hypomorphic SC development, deletion of a single Ptprb allele (creating Tek +/-; Ptprb NLS-LacZ/WT mice) rescued the SC development phenotype . This genetic interaction reveals that modulation of PTPRB activity can compensate for reduced TEK signaling, highlighting the delicate balance of phosphorylation-dependent signaling in vascular development. PTPRB antibodies are essential for validating genetic models, confirming protein expression levels, and investigating the molecular mechanisms underlying these developmental processes.

How can contradictory findings about PTPRB function be reconciled through experimental design?

Reconciling contradictory findings about PTPRB function requires sophisticated experimental design approaches that account for context-dependent effects and methodological variations. While the search results don't explicitly present contradictions, they highlight the multifaceted roles of PTPRB in different biological contexts. For instance, PTPRB promotes metastasis in colorectal cancer through EMT , yet it also plays crucial roles in vascular development through regulation of TEK phosphorylation . These diverse functions suggest potential context-dependent effects that researchers must carefully consider.

To address potential contradictions, researchers should implement comprehensive experimental designs that systematically evaluate PTPRB function across multiple models and conditions. This approach should include:

• Tissue and cell-type specificity analysis: Using PTPRB antibodies for comparative immunohistochemistry across different tissues and cell types can reveal context-dependent expression patterns. Combining this with functional assays specific to each tissue context can help elucidate why PTPRB may exhibit different roles in different settings.

• Dose-dependency studies: The search results indicate that partial reduction of PTPRB (heterozygosity) is sufficient to enhance TEK phosphorylation , suggesting that quantitative differences in PTPRB activity may lead to qualitatively different outcomes. Researchers should employ partial knockdown, heterozygous models, and complete knockout approaches to systematically evaluate dose-dependent effects.

• Temporal dynamics investigation: PTPRB's function may differ during development versus adult physiology or during different phases of disease progression. Time-course experiments using inducible systems can help resolve temporal aspects of PTPRB function.

• Pathway interaction mapping: Since PTPRB affects multiple substrates (TEK, VEGFR2, VE-Cadherin, FGD5) , researchers should employ phospho-proteomics approaches combined with PTPRB manipulation to comprehensively map its impact on signaling networks in different contexts. This systems-level analysis can help identify which downstream pathways predominate in specific biological settings.

What are common pitfalls when using PTPRB antibodies and how can they be avoided?

Working with PTPRB antibodies presents several potential challenges that researchers should anticipate and address to ensure reliable results. One common pitfall involves non-specific binding, particularly with polyclonal antibodies. To mitigate this issue, researchers should implement rigorous blocking protocols using 5-10% normal serum from the same species as the secondary antibody, combined with 1% BSA in PBS. Additionally, including appropriate negative controls such as isotype controls and antibody pre-absorption with immunizing peptide can help distinguish specific from non-specific signals .

Another significant challenge relates to detecting the correct PTPRB band in Western blotting applications. The discrepancy between observed (111 kDa) and calculated (224,301 Da) molecular weights can lead to uncertainty in band identification. Researchers should validate bands using positive controls known to express PTPRB and through knockdown or knockout approaches to confirm specificity. Furthermore, optimization of protein extraction methods is crucial, especially for membrane-associated proteins like PTPRB, with protocols incorporating mild detergents that preserve membrane protein integrity yielding better results.

Variable antibody performance between lots represents another common issue. Researchers should thoroughly validate each new antibody lot against previously characterized lots using consistent positive controls. Additionally, storage conditions significantly impact antibody performance—PTPRB antibodies should be stored at -20°C for long-term storage, with aliquoting recommended to avoid repeated freeze-thaw cycles. For frequent use, short-term storage at 4°C (up to one month) is acceptable . Finally, researchers should be aware that different applications may require different antibody concentrations; for instance, Western blotting typically requires dilutions of 1:500-2000, while ELISA applications may require more dilute solutions (1:5000-20000) . Titration experiments should be conducted for each specific application to determine optimal working concentrations.

How can researchers validate the specificity of PTPRB antibodies in their experimental systems?

Validating PTPRB antibody specificity requires a multi-faceted approach to ensure reliable and reproducible research outcomes. Researchers should implement a comprehensive validation strategy that combines multiple complementary techniques. Genetic approaches represent the gold standard for antibody validation—using PTPRB knockdown or knockout systems provides definitive evidence of antibody specificity. The search results describe a Ptprb NLS-LacZ knock-in reporter allele that prevents production of PTPRB protein , which could serve as an excellent negative control for antibody validation. Researchers can compare antibody signals between wild-type and Ptprb-deficient samples across multiple applications to confirm specificity.

Complementary approaches include peptide competition assays, where pre-incubation of the antibody with the immunizing peptide should abolish specific binding. This technique is particularly valuable for confirming the specificity of signals in immunohistochemistry and immunofluorescence applications. Another effective validation method involves testing the antibody across multiple applications—consistent results across Western blotting, immunoprecipitation, and immunohistochemistry provide stronger evidence for specificity than validation in a single application.

For advanced validation, researchers can employ orthogonal approaches such as mass spectrometry analysis of immunoprecipitated proteins or RNA-protein correlation studies. The latter approach compares protein detection by the antibody with mRNA expression levels of PTPRB across multiple cell types or tissues, with concordant patterns supporting antibody specificity. Finally, cross-validation using multiple antibodies targeting different epitopes of PTPRB can further strengthen confidence in experimental results—concordant results with antibodies recognizing distinct regions (such as those targeting AA 1655-1918 versus AA 280-360) provide compelling evidence for specific PTPRB detection.

What methods can be used to quantify PTPRB expression levels in research models?

Accurate quantification of PTPRB expression levels is essential for understanding its biological roles and regulatory mechanisms. Researchers can employ several complementary methodologies to achieve reliable quantification across different experimental contexts. Western blotting represents a widely used approach for semi-quantitative analysis of PTPRB protein levels. For optimal results, researchers should use recombinant PTPRB standards of known concentration to generate standard curves, allowing for more precise quantification. Normalization to housekeeping proteins is essential, and digital imaging systems with linear dynamic range should be employed for densitometric analysis .

For more sensitive and precise quantification, enzyme-linked immunosorbent assay (ELISA) techniques can be implemented. The search results indicate that many PTPRB antibodies are validated for ELISA applications, with recommended dilutions of 1:5000-20000 . Researchers can develop sandwich ELISA assays using capture and detection antibodies targeting different epitopes of PTPRB, enabling absolute quantification with potentially greater sensitivity than Western blotting. For tissue samples, quantitative immunohistochemistry offers valuable insights into both expression levels and spatial distribution. This approach involves standardized staining protocols, digital image acquisition, and computational analysis to quantify staining intensity and distribution patterns.

At the transcript level, quantitative real-time PCR (qRT-PCR) and RNA sequencing provide complementary data on PTPRB mRNA expression. While not directly measuring protein levels, these techniques offer valuable information on transcriptional regulation and can be correlated with protein quantification to provide a more comprehensive understanding of PTPRB expression. For single-cell or subcellular resolution, flow cytometry and imaging flow cytometry using fluorescently labeled PTPRB antibodies enable quantification of PTPRB expression at the cellular level with statistical power derived from analyzing large cell populations.

How might PTPRB inhibition be exploited as a therapeutic strategy in disease models?

PTPRB inhibition presents promising therapeutic potential based on its involvement in critical signaling pathways relevant to disease processes. The search results provide compelling evidence for two potential therapeutic applications: targeting PTPRB in cancer and in vascular development disorders. In colorectal cancer, research has demonstrated that PTPRB promotes metastasis via inducing epithelial-mesenchymal transition (EMT) . PTPRB knockdown decreased the number of invasive CRC cells in vitro and reduced tumor metastasis in vivo, suggesting that PTPRB inhibition could represent an effective strategy for preventing or reducing metastatic spread in colorectal cancer patients .

In vascular contexts, genetic studies have shown that reducing PTPRB expression by deleting a single allele can enhance TEK phosphorylation and rescue vascular development defects in Tek haploinsufficient mice . This finding suggests that partial inhibition of PTPRB might be sufficient to achieve therapeutic benefits while minimizing potential adverse effects. Importantly, heterozygous Ptprb NLS-LacZ/WT mice are born normally and exhibit normal Schlemm's canal development, indicating that partial reduction in PTPRB activity is well-tolerated during development .

For therapeutic development, researchers could pursue several approaches. Small molecule inhibitors targeting PTPRB phosphatase activity represent one promising strategy. Alternatively, antisense oligonucleotides, siRNAs, or CRISPR-based approaches could reduce PTPRB expression levels. Monoclonal antibodies that interfere with PTPRB function or substrate interactions could provide another avenue for therapeutic development. The development of such therapeutics would require careful validation using PTPRB antibodies to confirm target engagement and efficacy in preclinical models before advancing to clinical testing.

What novel techniques are emerging for studying PTPRB interactions with its binding partners?

Emerging techniques for studying PTPRB interactions with binding partners are expanding our understanding of this phosphatase's complex regulatory networks. While the search results don't explicitly detail novel techniques, several cutting-edge approaches are particularly well-suited for investigating PTPRB interactions. Proximity labeling methods such as BioID or APEX2 represent powerful approaches for identifying protein interactions in living cells. By fusing these enzymes to PTPRB, researchers can biotinylate proteins in close proximity, followed by streptavidin pulldown and mass spectrometry to identify the interaction network. This approach is particularly valuable for studying membrane proteins like PTPRB, where traditional co-immunoprecipitation methods may face challenges with membrane solubilization.

Förster resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) enable real-time visualization of protein interactions in living cells. By tagging PTPRB and potential binding partners with appropriate fluorophores or bioluminescent proteins, researchers can monitor interactions and their dynamics in response to stimuli or perturbations. Cryo-electron microscopy (cryo-EM) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) provide structural insights into protein complexes. These techniques could reveal how PTPRB interacts with substrates like TEK, VE-Cadherin, VEGFR2, and FGD5 , potentially identifying structural features that could be targeted therapeutically.

Additionally, advanced genetic approaches such as CRISPR-based screening can identify genetic dependencies and synthetic lethal interactions involving PTPRB. By combining CRISPR screens with phenotypic assays relevant to PTPRB function (such as cell migration, angiogenesis, or EMT), researchers can uncover novel genetic interactions. Validation of these interactions would rely heavily on PTPRB antibodies for confirming protein expression and localization patterns. Single-cell approaches, including single-cell RNA-seq combined with protein detection methods, could provide insights into how PTPRB interactions vary across cell populations and states, revealing context-dependent regulatory mechanisms.

How does the role of PTPRB vary across different types of cancer and vascular disorders?

PTPRB exhibits context-dependent functions across different cancer types and vascular disorders, reflecting the complexity of phosphatase signaling networks. While the search results primarily focus on colorectal cancer and vascular development in the eye, broader implications can be inferred. In colorectal cancer, PTPRB promotes metastasis through inducing epithelial-mesenchymal transition (EMT) . PTPRB is expressed at significantly higher levels in CRC tissues compared to adjacent non-tumor tissues and in CRC cell lines with high invasion potential . This suggests that in CRC, PTPRB functions as a pro-metastatic factor by promoting cellular plasticity and invasive behavior.