PTPRO Antibody

What is PTPRO and what are its structural and functional characteristics?

PTPRO is a 138.3 kDa protein that belongs to the protein tyrosine phosphatase family. It possesses a distinctive structure consisting of:

• A large extracellular domain containing eight fibronectin type III-like repeats

• A hydrophobic transmembrane segment

• A single intracellular PTPase domain

PTPRO plays a critical role in regulating the glomerular pressure/filtration rate relationship through effects on podocyte structure and function. It maintains the integrity of the glomerular filtration barrier by regulating tyrosine phosphorylation of podocyte proteins. Research indicates that PTPRO knockout mice exhibit altered podocyte morphology, with changes in primary podocyte processes and decreased length of interdigitating tertiary processes and slit-diaphragms between them .

What types of PTPRO antibodies are available for research applications?

Multiple types of PTPRO antibodies are available for research:

Specific examples include:

• mAb 4C3: Binds to amino acid core of PTPro (fibronectin repeat 3), affects phosphatase activity

• mAb P8E7: Recognizes conformational epitope, does not affect phosphatase activity

• mAbs 1B4 and 1D1: Bind to rat PTPRO in IgG2a isotype

• Polyclonal antibodies against the extracellular domain (ECD)

How do epitope-specific PTPRO antibodies affect experimental outcomes?

Different PTPRO antibodies recognize distinct epitopes and can produce varying experimental results:

• Fibronectin domain targeting: mAb 4C3, which binds to fibronectin repeat 3, decreases PTPRO phosphatase activity and increases glomerular albumin permeability (P₍alb₎) in a time and concentration-dependent manner

• Conformational epitope recognition: mAb P8E7 recognizes a conformational epitope that is destroyed by denaturation and does not affect phosphatase activity or glomerular permeability

• Functional effects: Some antibodies binding to the ECD can acutely increase P₍alb₎, potentially through inactivation of phosphatase activity

• Species specificity: Antibodies developed against rabbit PTPRO (e.g., 4C3) may not bind to rat PTPRO, while species-specific antibodies show consistent binding and functional effects

These differences highlight the importance of selecting appropriate antibodies based on the specific research question and experimental design.

What are the optimal conditions for detecting PTPRO by Western blot?

Detecting PTPRO by Western blot requires specific conditions due to its high molecular weight and potential for dimerization:

• Sample preparation: Triton X-100-containing Tris buffer is effective for extraction from tissues

• Molecular weight patterns:

  • Under reducing conditions: PTPRO appears as a smear from 180-220 kDa

  • Under non-reducing conditions: A band appears around 350 kDa, likely representing the dimeric form of full-length PTPRO

• Recommended dilutions: Typically 1:500-1:2000, though optimization may be necessary

• Buffer conditions: PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 is commonly used for antibody storage

• Storage: Store at -20°C for long-term; for frequent use, 4°C storage for up to one month is acceptable

Researchers should validate the specific molecular weight pattern in their system, as it may vary depending on tissue source and extraction method.

How can researchers validate the specificity of PTPRO antibodies?

Rigorous validation of PTPRO antibodies should include:

• Knockout/knockdown controls: Testing in PTPRO⁻/⁻ mice tissues or CRISPR-Cas9 modified cell lines to establish baseline signals and identify non-specific binding

• Peptide competition assays: Pre-incubation of antibody with specific antigen (e.g., PTPro ECD GST fusion protein) should abolish signal

• Multi-antibody approach: Comparing results from different antibodies targeting different PTPRO epitopes

• Western blot analysis: Confirming specificity through expected molecular weight patterns

• Enhanced validation techniques: Orthogonal RNAseq validation can provide additional confidence

• Functional assays: For antibodies claimed to affect PTPRO activity, phosphatase activity assays using p-nitrophenylphosphate as a substrate

Documentation of validation experiments is crucial for reliable interpretation of results.

What functional effects do anti-PTPRO antibodies have on phosphatase activity?

Several anti-PTPRO antibodies have demonstrated significant effects on PTPRO phosphatase activity:

• Inhibitory effects: mAb 4C3 decreases phosphatase activity when incubated with glomeruli, correlating with increased glomerular permeability

• No effect: mAb P8E7 does not diminish phosphatase activity and does not alter permeability, despite binding to PTPRO

• Experimental assessment: Phosphatase activity can be measured after immunoprecipitation using p-nitrophenylphosphate as a substrate

• Physiological implications: Tonic PTPro activity appears to be required to maintain normal characteristics of the glomerular filtration barrier

These findings suggest that antibody-mediated modulation of PTPRO activity may have significant physiological consequences and potential therapeutic applications.

How do PTPRO antibodies perform in immunohistochemistry applications?

Performance in immunohistochemistry varies by antibody and requires optimization:

• Antigen retrieval methods: TE buffer pH 9.0 or alternatively citrate buffer pH 6.0 is recommended

• Dilution ranges: Typical dilutions for IHC range from 1:50-1:4000, depending on the specific antibody

• Tissue fixation: Formalin/PFA-fixed paraffin-embedded tissues are commonly used

• Signal localization: In kidney tissue, PTPRO antibodies typically show localization to podocytes at the glomerular filtration barrier

• Multiplex capabilities: Some PTPRO antibodies (e.g., ab322047) have been validated for multiplex IHC with other markers like SLC5A2 and Aquaporin 3

• Species reactivity: Carefully check reactivity with human, mouse, rat, or other species of interest

Each antibody may require specific optimization depending on the tissue source and fixation method.

What methodological considerations are important when studying PTPRO in kidney disease models?

When investigating PTPRO in kidney disease:

• Isolated glomeruli preparation: Cold perfusion and iron embolization techniques can be used to isolate glomeruli for functional studies

• Permeability assays: The glomerular albumin permeability (P₍alb₎) assay provides a sensitive measurement of glomerular dysfunction before detectable proteinuria

• Antibody incubation conditions: Time (10-120 minutes) and concentration (5-100 μg/ml) should be optimized based on the specific antibody

• Control antibodies: Include isotype-matched irrelevant antibodies that do not bind to glomeruli or target unrelated proteins

• Species considerations: Results may vary between rabbit and rat glomeruli, necessitating species-specific antibodies and controls

• Correlation with in vivo models: Changes in P₍alb₎ often precede detectable proteinuria in various kidney disease models

These methodological considerations ensure reliable and reproducible results when studying PTPRO's role in kidney physiology and pathology.

How is PTPRO implicated in cancer research, and what experimental approaches are used?

PTPRO has emerging roles in cancer biology:

• Anti-proliferation assays: PTPRO inhibition using compounds like GP03 suppresses the proliferative abilities of tumor cells in pancreatic, blood, and breast cancers

• Sulforhodamine B assay: Used to assess cellular proliferation after treatment with PTPRO inhibitors

• Expression analysis: Immunohistochemical detection of PTPRO expression in tumor versus normal tissues can be correlated with clinical outcomes

• ERBB2-positive breast cancer: PTPRO deficiency contributes to poor prognosis and lapatinib resistance

• Hepatocellular carcinoma: PTPRO-mediated autophagy prevents hepatosteatosis and tumorigenesis; PTPRO maintains T cell immunity in the tumor microenvironment

These approaches highlight PTPRO as both a potential therapeutic target and prognostic marker in various cancers.

What is known about PTPRO's role in neurological conditions?

Recent research has revealed PTPRO's significance in neurological function:

• Age-related decline: Hippocampal PTPRO levels decline with age, potentially affecting cognitive function

• Chemotherapy-related cognitive impairment (CRCI): PTPRO deficiency leads to CRCI-relevant cognitive impairment in mice models

• Behavioral assessment: Y-maze and Morris water maze (MWM) tests can evaluate spatial learning and memory abilities in PTPRO knockout or deficient models

• Restoration experiments: Intrahippocampal injection of lentiviral PTPRO into the CA3 region significantly rescues learning and memory abilities in PTPRO-deficient mice

• Therapeutic targeting: Compounds like berberine (BBR) can be repurposed to target PTPRO-deficient states, suggesting potential preventive strategies against CRCI development

These findings suggest PTPRO as a potential therapeutic target for cognitive impairment, particularly in aging and cancer treatment contexts.

How should researchers design experiments to study PTPRO interactions with other proteins?

To investigate PTPRO's protein interactions:

• Co-immunoprecipitation: Glomerular extracts can be incubated with antibodies and protein G beads to isolate PTPRO and associated proteins

• Western blot analysis: Following immunoprecipitation, samples can be analyzed by Western blotting to identify co-precipitated proteins

• Functional assessment: Phosphatase activity of immunoprecipitated complexes can be measured to determine if interactions enhance or inhibit activity

• Controls: Include isotype-matched control antibodies to identify non-specific binding

• Fusion proteins: GST fusion proteins containing specific domains of PTPRO can be used to map interaction regions

• Cross-linking approaches: May be necessary to capture transient interactions

These methods allow for comprehensive characterization of PTPRO's interactome in different physiological and pathological contexts.

What are the latest advances in PTPRO inhibitor development and application?

Recent developments in PTPRO inhibition include:

• Small molecule inhibitors: Compounds like GP03 have been developed and tested in cancer models

• Anti-proliferative effects: PTPRO inhibition has demonstrated anti-proliferative activities in various cancer cell types including pancreatic, blood, and breast cancer

• Therapeutic vulnerability: PTPRO deficiency in specific contexts (e.g., ERBB2-positive breast cancer) confers therapeutic vulnerability that can be targeted

• Berberine repurposing: BBR has shown promise in targeting PTPRO-deficient states, particularly in neurological contexts

• Assessment methods: Techniques such as the sulforhodamine B assay are used to evaluate cellular responses to PTPRO inhibition

These advances highlight the potential of PTPRO as a therapeutic target in multiple disease contexts.