PTPRE Antibody

What is PTPRE and what cellular functions does it perform?

PTPRE is a receptor-type protein tyrosine phosphatase belonging to the protein-tyrosine phosphatase family. In humans, the canonical protein consists of 700 amino acid residues with a molecular mass of approximately 80.6 kDa. PTPRE is primarily localized in the cell membrane and cytoplasm, where it functions in protein dephosphorylation pathways . The protein plays important roles in signal transduction, cell differentiation, and metabolism regulation. PTPRE has been reported to be expressed in giant cell tumor (osteoclastoma rich in multinucleated osteoclastic cells), suggesting its potential involvement in bone metabolism and pathological conditions .

What are the common applications for PTPRE antibodies in research?

PTPRE antibodies are versatile tools employed across multiple experimental techniques. The most widely used application is Western Blotting (WB), which allows researchers to detect and quantify PTPRE protein expression in cellular extracts . Additionally, these antibodies are frequently used in Immunohistochemistry (IHC) to visualize the spatial distribution of PTPRE in tissue sections, Flow Cytometry to analyze PTPRE expression at the single-cell level, Immunofluorescence (IF) for subcellular localization studies, and Enzyme-Linked Immunosorbent Assay (ELISA) for quantitative protein detection . The selection of the appropriate application depends on the specific research question and experimental design.

How do I choose between polyclonal and monoclonal PTPRE antibodies?

The choice between polyclonal and monoclonal PTPRE antibodies depends on your experimental requirements:

For initial characterization, polyclonal antibodies may be preferable due to their ability to recognize multiple epitopes, while monoclonal antibodies like clone 4B2 are more suitable for experiments requiring high specificity and reproducibility .

What are the optimal conditions for Western blotting with PTPRE antibodies?

For optimal Western blotting results with PTPRE antibodies, consider the following protocol:

• Sample preparation: Extract proteins using RIPA or NP-40 buffer containing protease and phosphatase inhibitors.

• Protein loading: Load 20-50 μg of total protein per lane.

• Gel separation: Use 8-10% SDS-PAGE gels to properly resolve the ~80.6 kDa PTPRE protein.

• Transfer conditions: Transfer proteins to PVDF membrane at 100V for 90 minutes in cold transfer buffer containing 20% methanol.

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

• Primary antibody incubation: Dilute PTPRE antibody (typically 1:500-1:2000, depending on the specific product) in blocking solution and incubate overnight at 4°C.

• Secondary antibody: Use appropriate HRP-conjugated secondary antibody (anti-rabbit or anti-mouse, depending on the primary antibody host) at 1:5000-1:10000 dilution.

• Detection: Use enhanced chemiluminescence (ECL) detection system with exposure times ranging from 30 seconds to 5 minutes.

If targeting specific PTPRE domains, select antibodies recognizing appropriate amino acid regions, such as those targeting AA 511-600 for C-terminal studies or AA 20-200 for N-terminal studies .

How can I optimize immunohistochemistry protocols for PTPRE detection in tissue samples?

For effective immunohistochemical detection of PTPRE in tissue samples:

• Fixation: Use 10% neutral buffered formalin for 24-48 hours, followed by paraffin embedding.

• Sectioning: Cut 4-6 μm sections and mount on positively charged slides.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes.

• Endogenous peroxidase blocking: Incubate sections with 3% hydrogen peroxide for 10 minutes.

• Protein blocking: Block with 5% normal serum from the same species as the secondary antibody for 1 hour.

• Primary antibody: Incubate with PTPRE antibody (typically 1:100-1:500 dilution) overnight at 4°C.

• Detection system: Use biotin-streptavidin or polymer-based detection systems for enhanced sensitivity.

• Counterstaining: Counterstain with hematoxylin for nuclear visualization.

• Controls: Always include positive and negative controls to validate staining specificity.

For frozen sections, fix briefly in cold acetone or 4% paraformaldehyde prior to immunostaining and adjust antibody dilutions accordingly. Some PTPRE antibodies have been specifically validated for IHC applications, including those targeting the cytoplasmic domain, which may provide superior results in tissue staining applications .

What cross-reactivity considerations should I address when selecting PTPRE antibodies?

Cross-reactivity is a critical consideration when selecting PTPRE antibodies. Many PTPRE antibodies demonstrate reactivity across multiple species, which can be advantageous for comparative studies but requires careful validation. Based on the available products, consider the following cross-reactivity patterns:

To ensure specificity, validate antibodies using positive and negative controls, including PTPRE knockout or knockdown samples. Additionally, pre-absorption controls with the immunizing peptide can help confirm specificity . Some antibodies have been specifically purified by antigen affinity methods to reduce non-specific binding.

How can I investigate PTPRE phosphorylation states and their functional significance?

Investigating PTPRE phosphorylation states requires a multi-faceted approach:

• Phospho-specific antibodies: If available, use antibodies that specifically recognize phosphorylated PTPRE at known regulatory sites. Alternatively, use anti-phosphotyrosine antibodies following PTPRE immunoprecipitation.

• Phosphatase assays: Measure PTPRE enzymatic activity using:

  • In vitro assays with purified PTPRE and synthetic phosphopeptide substrates

  • Para-nitrophenyl phosphate (pNPP) assays for general phosphatase activity

  • 32P-labeled substrates for higher sensitivity quantification

• Mutagenesis studies: Generate phospho-mimetic (e.g., Tyr→Glu) or phospho-deficient (e.g., Tyr→Phe) PTPRE mutants to functionally assess the impact of phosphorylation at specific residues.

• Correlation with cellular functions: After treatments that modulate PTPRE phosphorylation, analyze:

  • Changes in substrate phosphorylation using phospho-specific antibodies

  • Alterations in protein-protein interactions via co-immunoprecipitation

  • Subcellular localization shifts using fractionation or immunofluorescence

  • Functional outcomes through cell-based assays (migration, proliferation, etc.)

• Mass spectrometry: For unbiased identification of phosphorylation sites, immunoprecipitate PTPRE and analyze by LC-MS/MS.

This integrative approach allows researchers to establish connections between PTPRE phosphorylation status and its functional roles in signaling pathways.

What strategies can resolve contradictory PTPRE expression data between different antibodies?

When facing contradictory PTPRE expression data between different antibodies, implement the following systematic troubleshooting approach:

• Epitope mapping: Confirm the exact epitopes recognized by each antibody. Different antibodies targeting distinct domains may yield different results if:

  • Certain epitopes are masked by protein-protein interactions

  • Post-translational modifications affect antibody binding

  • Protein conformation varies between experimental conditions

  • Alternative splicing generates isoform-specific epitopes

• Validation with orthogonal methods:

  • Complement antibody-based detection with mRNA quantification (RT-qPCR)

  • Use CRISPR/Cas9 or siRNA knockdown controls to confirm specificity

  • Overexpress tagged PTPRE to verify antibody recognition

• Systematic comparison of experimental conditions:

  • Standardize sample preparation (lysis buffers, protease inhibitors)

  • Test multiple blocking agents to reduce non-specific binding

  • Optimize antibody concentrations with titration experiments

  • Evaluate multiple detection systems (chemiluminescence vs. fluorescence)

• Statistical analysis: Perform replicate experiments and apply appropriate statistical tests to determine if differences are significant or within expected experimental variation.

By implementing this comprehensive approach, researchers can reconcile contradictory data and establish reliable PTPRE expression profiles across experimental systems.

How can I distinguish between PTPRE isoforms and study their differential functions?

PTPRE exists in multiple isoforms with distinct subcellular localizations and potentially different functions. To distinguish between these isoforms:

• Isoform-specific antibodies: Select antibodies targeting unique regions of specific PTPRE isoforms. For instance:

  • Antibodies against the transmembrane domain specifically detect receptor PTPRE

  • Antibodies against cytoplasmic PTPRE-specific sequences detect non-receptor isoforms

• Molecular weight discrimination:

  • Receptor PTPRE: ~80.6 kDa (full-length)

  • Cytoplasmic PTPRE: ~72 kDa

• Subcellular fractionation: Separate cellular compartments (membrane, cytosol, nucleus) before immunoblotting to enrich for specific isoforms based on their localization patterns.

• Isoform-specific functional assays:

  Isoform	Subcellular Localization	Suggested Functional Assays
  Receptor PTPRE	Cell membrane	- Membrane-impermeable substrate assays - Cell surface biotinylation - Receptor clustering studies
  Cytoplasmic PTPRE	Cytoplasm	- Cytosolic substrate dephosphorylation - Interactions with cytoskeletal components - Nuclear translocation studies

• Isoform-specific expression: Use isoform-specific qPCR primers to quantify mRNA expression levels of different isoforms across tissues or experimental conditions.

• CRISPR/Cas9 gene editing: Design guide RNAs targeting isoform-specific exons to create isoform-selective knockout cell lines for comparative functional studies.

This multi-modal approach enables comprehensive analysis of PTPRE isoform-specific expression patterns and functions.

How can I address non-specific binding or high background issues in PTPRE immunodetection?

Non-specific binding and high background are common challenges in immunodetection experiments. For PTPRE-specific troubleshooting:

• Antibody validation and selection:

  • Test multiple PTPRE antibodies from different suppliers

  • Select antibodies that have been affinity-purified against the specific antigen

  • Verify antibody specificity using PTPRE knockout or knockdown samples

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Increase blocking time (2-3 hours at room temperature or overnight at 4°C)

  • Add 0.1-0.3% Triton X-100 to blocking solution to reduce hydrophobic interactions

• Antibody dilution and incubation:

  • Titrate primary antibody concentration (typically 1:500-1:2000 for Western blots)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Add 0.05-0.1% Tween-20 to antibody diluent

• Washing optimization:

  • Increase washing duration and frequency (5-6 washes of 10 minutes each)

  • Add higher concentrations of detergent (0.1-0.2% Tween-20) to wash buffer

  • Use automated washers for consistent and thorough washing

• Application-specific adaptations:

  Application	Specific Troubleshooting Strategies
  Western Blot	- Use PVDF instead of nitrocellulose membrane - Increase SDS concentration in transfer buffer - Apply gradient gels for better resolution
  IHC	- Optimize antigen retrieval (pH, duration, temperature) - Use biotin-free detection systems - Apply Sudan Black B to reduce autofluorescence
  IF	- Include 0.1-0.3% Triton X-100 for cell permeabilization - Use image-based quantification to subtract background - Apply signal enhancement systems (tyramide)
  Flow Cytometry	- Include viability dye to exclude dead cells - Use Fc receptor blocking reagents - Optimize fixation and permeabilization buffers

By systematically implementing these strategies, researchers can significantly reduce background issues in PTPRE immunodetection experiments.

What factors affect PTPRE antibody sensitivity and how can I improve detection of low-abundance PTPRE?

Detecting low-abundance PTPRE requires optimizing multiple experimental parameters:

• Sample enrichment strategies:

  • Immunoprecipitate PTPRE before detection to concentrate the target protein

  • Use subcellular fractionation to enrich for compartments where PTPRE is localized

  • Apply phosphatase inhibitors during sample preparation to preserve phosphorylated forms

• Signal amplification methods:

  • For Western blots: Use high-sensitivity ECL substrates or fluorescent detection systems

  • For IHC/IF: Apply tyramide signal amplification (TSA) or polymer-based detection systems

  • For flow cytometry: Utilize multi-layer detection with biotin-streptavidin systems

• Instrumentation optimization:

  • Use highly sensitive CCD cameras for Western blot imaging

  • Apply confocal or super-resolution microscopy for IF detection

  • Optimize PMT voltage settings for flow cytometry

• Buffer and reagent considerations:

  • Add protease and phosphatase inhibitor cocktails to lysis buffers

  • Use freshly prepared reducing agents in sample buffers

  • Apply antigen retrieval optimization for fixed tissue samples

• Antibody selection for low-abundance detection:

  Antibody Type	Sensitivity Characteristics
  High-affinity monoclonals	Lower detection threshold but epitope-restricted
  Pooled polyclonals	Enhanced signal through multiple epitope recognition
  Directly conjugated antibodies	Elimination of secondary antibody background
  Recombinant antibodies	Consistent lot-to-lot performance for quantitative studies

• Loading controls and normalization:

  • Use appropriate housekeeping proteins based on experimental conditions

  • Apply total protein normalization (stain-free gels, Ponceau S) for more accurate quantification

  • Include positive controls with known PTPRE expression levels

These comprehensive approaches can significantly enhance the detection of low-abundance PTPRE in various experimental systems.

How can I develop co-localization studies to investigate PTPRE interactions with potential binding partners?

Co-localization studies provide valuable insights into PTPRE's spatial relationships with interacting proteins. Implement the following methodological approach:

• Dual immunofluorescence labeling:

  • Select antibodies from different host species (e.g., rabbit anti-PTPRE and mouse anti-partner protein)

  • Use fluorophore-conjugated secondary antibodies with well-separated emission spectra

  • Include single-label controls to verify antibody specificity and channel bleed-through

• Advanced microscopy techniques:

  • Confocal microscopy for improved spatial resolution

  • Super-resolution microscopy (STED, PALM, STORM) for nanoscale co-localization

  • Live-cell imaging with fluorescent protein fusions to track dynamic interactions

• Quantitative co-localization analysis:

  • Calculate Pearson's correlation coefficient (PCC) or Mander's overlap coefficient

  • Apply intensity correlation analysis (ICA) for more robust assessment

  • Use specialized software (ImageJ with Coloc2, CellProfiler) for unbiased quantification

• Proximity ligation assay (PLA):

  • Detect protein-protein interactions within 40 nm proximity

  • Generate amplified fluorescent signals only when proteins are closely associated

  • Quantify interaction events as discrete fluorescent spots

• Controls and validation:

  • Positive controls using known interaction partners

  • Negative controls with proteins known not to interact with PTPRE

  • Functional validation through mutagenesis of interaction domains

This systematic approach provides both qualitative and quantitative assessment of PTPRE's spatial relationships with potential binding partners.

What approaches can I use to study PTPRE in primary cells and tissue specimens?

Studying PTPRE in primary cells and tissues presents unique challenges that require specialized techniques:

• Tissue-specific expression profiling:

  • Immunohistochemistry on tissue microarrays (TMAs) to compare expression across multiple samples

  • Single-cell RNA-seq to identify cell populations expressing PTPRE

  • Laser capture microdissection combined with Western blotting or qPCR for region-specific analysis

• Primary cell isolation and culture:

  • Isolate primary cells expressing PTPRE (e.g., osteoclasts from giant cell tumors)

  • Optimize fixation protocols to preserve PTPRE epitopes

  • Adapt lysis procedures for limited sample amounts

• Ex vivo functional studies:

  • Tissue slice cultures to maintain PTPRE in its native microenvironment

  • Organoid models to study PTPRE in 3D tissue-like structures

  • Patient-derived xenografts (PDX) for in vivo functional studies

• Multiplex detection approaches:

  • Multiplex immunofluorescence to co-stain PTPRE with cell type markers

  • Imaging mass cytometry for simultaneous detection of dozens of proteins

  • Spatial transcriptomics to correlate PTPRE protein localization with gene expression

• Tissue-specific considerations:

  Tissue/Cell Type	Special Considerations
  Bone/Osteoclasts	- Decalcification affects epitope preservation - TRAP co-staining to identify osteoclasts
  Frozen tissues	- Brief fixation in 4% PFA to preserve morphology - Optimization of permeabilization
  FFPE tissues	- Extended antigen retrieval - Signal amplification systems
  Primary immune cells	- Non-enzymatic isolation methods - Minimizing activation during processing

These approaches enable comprehensive characterization of PTPRE expression and function in physiologically relevant primary cells and tissues.

How can I design experiments to assess PTPRE's role in disease models and potential therapeutic targeting?

Investigating PTPRE's role in disease contexts requires comprehensive experimental designs:

• Expression correlation with disease progression:

  • Analyze PTPRE levels across disease stages using tissue microarrays

  • Correlate expression with clinical outcomes and prognostic indicators

  • Examine isoform-specific expression patterns in normal versus diseased tissues

• Genetic manipulation in disease models:

  • CRISPR/Cas9 knockout or knockin of PTPRE in cell-based disease models

  • Conditional knockout mice to study tissue-specific PTPRE functions

  • Viral vector-mediated overexpression or silencing in animal models

• Pharmacological modulation:

  • Small molecule inhibitors targeting PTPRE catalytic activity

  • Allosteric modulators affecting PTPRE conformation or interactions

  • Peptide-based approaches to disrupt specific protein-protein interactions

• Disease-specific functional readouts:

  Disease Context	Functional Assays
  Cancer	- Cell proliferation and apoptosis assays - Invasion and migration studies - Tumor formation in xenograft models
  Inflammatory conditions	- Cytokine production profiles - Leukocyte adhesion and migration - NF-κB pathway activation
  Metabolic disorders	- Insulin signaling cascade analysis - Glucose uptake measurements - Lipid metabolism assessment
  Bone disorders	- Osteoclast differentiation and function - Bone resorption assays - Micro-CT analysis of bone structure

• Therapeutic targeting validation:

  • Antibody-drug conjugates targeting cell-surface PTPRE

  • RNA interference approaches for isoform-specific knockdown

  • Structure-based drug design targeting PTPRE catalytic pocket or allosteric sites

By implementing these comprehensive experimental approaches, researchers can establish causal relationships between PTPRE function and disease pathogenesis, potentially identifying novel therapeutic strategies.