PTPRZ1 Antibody

What is PTPRZ1 and why is it relevant to neuroscience and cancer research?

PTPRZ1, also known as HTPZP2, PTPRZ, PTPRZ2, and PTPZ, belongs to the protein-tyrosine phosphatase family and Receptor class 5 subfamily. It plays a crucial role in the regulation of specific developmental processes in the central nervous system (CNS) . In recent years, PTPRZ1 has gained significant attention as a relevant marker of glioma stem cells, a population considered responsible for chemoresistance and radioresistance in glioblastoma . Its involvement in these critical biological processes makes it an important target for both basic neuroscience research and cancer therapeutics development.

What isoforms of PTPRZ1 exist and how can they be differentiated in experimental settings?

PTPRZ1 has multiple isoforms with varying molecular weights: 254 kDa, 163 kDa, and 90 kDa . When working with PTPRZ1 antibodies, researchers should be aware that the observed molecular weight in Western blot applications is typically 163-175 kDa, which may differ from the calculated molecular weight of 255 kDa . To differentiate between isoforms, researchers should use high-resolution gel electrophoresis techniques and potentially employ isoform-specific antibodies where available. Additionally, RNA analysis methods such as RT-PCR with isoform-specific primers can help distinguish between different transcript variants when protein-level differentiation is challenging.

Which model systems express PTPRZ1 and are suitable for antibody validation?

Based on reactivity data, PTPRZ1 antibodies have been validated in human, mouse, and rat samples . For cell culture models, HepG2 and HeLa cells have shown positive detection in Western blot applications . For tissue analysis, rat cerebellum tissue has demonstrated positive immunohistochemical detection . When establishing new model systems, researchers should first confirm PTPRZ1 expression using multiple detection methods (e.g., qPCR, Western blot) before proceeding with functional studies.

What are the optimal protocols for PTPRZ1 antibody use in Western blotting applications?

For Western blot applications, the recommended dilution range for PTPRZ1 antibodies is 1:500-1:1000 . Optimization is critical for each experimental system. A detailed protocol should include:

• Sample preparation: Cells or tissues should be lysed in RIPA buffer supplemented with protease inhibitors

• Protein quantification: BCA or Bradford assay to ensure equal loading

• Gel electrophoresis: 6-8% SDS-PAGE is recommended for optimal resolution of the high molecular weight PTPRZ1 isoforms

• Transfer: Wet transfer at low voltage (30V) overnight for efficient transfer of large proteins

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody incubation: Apply PTPRZ1 antibody at optimized dilution (start with 1:500) in blocking buffer overnight at 4°C

• Washing: 3-5 washes with TBST, 5 minutes each

• Secondary antibody incubation: HRP-conjugated anti-rabbit IgG at 1:5000 for 1 hour at room temperature

• Signal detection: ECL substrate followed by imaging

How should researchers optimize immunohistochemistry protocols for PTPRZ1 detection in brain tissue?

For immunohistochemistry applications, the recommended dilution range is 1:50-1:500 . When working with brain tissue, particularly for PTPRZ1 detection, consider the following optimization steps:

• Fixation: 4% paraformaldehyde fixation for 24-48 hours is typical for brain tissue

• Antigen retrieval: Use TE buffer pH 9.0 as suggested for optimal results; alternatively, citrate buffer pH 6.0 may be used

• Blocking: 10% normal serum (from secondary antibody host species) with 0.3% Triton X-100

• Primary antibody incubation: Apply PTPRZ1 antibody at 1:100 dilution (initial testing) overnight at 4°C

• Detection system: ABC method or polymer-based detection systems often provide the best signal-to-noise ratio

• Counterstaining: Light hematoxylin staining to visualize tissue architecture

• Controls: Include both positive controls (rat cerebellum) and negative controls (primary antibody omission)

How can researchers effectively use PTPRZ1 antibodies for immunofluorescence studies?

While specific dilution recommendations for immunofluorescence were not provided in the search results, published applications indicate successful use in IF applications . For optimal results:

• Begin with dilutions in the range of 1:100-1:200 based on IHC recommendations

• Use high-quality fluorescent secondary antibodies with minimal cross-reactivity

• Include appropriate controls to assess background fluorescence

• Consider dual staining with neuronal or glial markers to assess cell-type specificity

• For brain tissue sections, use a suitable antigen retrieval method as described for IHC

• For cultured cells, a mild permeabilization with 0.1-0.3% Triton X-100 is typically sufficient

How is PTPRZ1 implicated in glioblastoma pathogenesis and what are the experimental approaches to study this relationship?

PTPRZ1 has been identified as a marker of glioma stem cells, a population considered responsible for treatment resistance in glioblastoma . To investigate the functional role of PTPRZ1 in glioblastoma:

• Expression analysis: Compare PTPRZ1 expression levels between glioblastoma tissues/cells and normal brain tissue using immunohistochemistry, Western blotting, and qPCR

• Functional knockdown/knockout studies: Use siRNA, shRNA, or CRISPR-Cas9 approaches to suppress PTPRZ1 expression

• Pathway analysis: Examine downstream signaling pathways affected by PTPRZ1 manipulation, particularly focusing on ERK phosphorylation

• Phenotypic assays: Assess changes in cell proliferation, migration, invasion, and stem cell properties following PTPRZ1 modulation

• In vivo models: Evaluate tumor growth and invasion in orthotopic xenograft models with PTPRZ1 knockdown or overexpression

Research has shown that suppression of PTPRZ1 by siRNA inhibits glioblastoma growth both in vitro and in vivo, highlighting its potential as a therapeutic target .

What methodological approaches can be used to target PTPRZ1 in glioblastoma therapy research?

Several approaches have been developed to target PTPRZ1 in experimental glioblastoma therapy:

• Monoclonal antibodies: Anti-PTPRZ1 monoclonal antibodies, such as clone 2F10, have been shown to impede PTN-mediated processes essential for glioma stem cell self-renewal, migration, and invasion

• CAR T-cell therapy: PTPRZ1-targeting chimeric antigen receptor (CAR) T cells have demonstrated antigen-specific cytotoxicity against glioblastoma cells and delayed tumor growth in orthotopic xenograft models

• RNA interference: siRNA or shRNA targeting PTPRZ1 can be used to suppress its expression and evaluate effects on tumor growth

• Small molecule inhibitors: While not explicitly mentioned in the search results, researchers can explore small molecule inhibitors of PTPRZ1 phosphatase activity

• Gene editing: CRISPR-Cas9 technology can be employed to knockout PTPRZ1 in glioma stem cells to assess its role in tumorigenesis

How do researchers evaluate the efficacy of PTPRZ1-targeting approaches in preclinical models?

When assessing the effectiveness of PTPRZ1-targeting strategies in preclinical models, researchers typically employ the following methods:

• In vitro assays:

  • Clonogenic assays to measure self-renewal capacity

  • Transwell migration and invasion assays to assess motility and invasiveness

  • Cell viability and apoptosis assays to determine cytotoxic effects

  • Sphere formation assays to evaluate cancer stem cell properties

• In vivo models:

  • Orthotopic xenograft tumor models in immunodeficient mice (e.g., NOD/SCIDγ mice)

  • Bioluminescence imaging to monitor tumor growth longitudinally

  • Survival analysis to determine therapeutic benefit

  • Histopathological examination of tumor tissue to assess proliferation, apoptosis, and invasion

• Molecular analysis:

  • Western blotting to measure changes in ERK phosphorylation and other downstream pathways

  • Immunohistochemistry to assess target engagement and pathway modulation in tumor tissue

  • RNA-seq or proteomics to evaluate global changes in gene expression or protein profiles

How is PTPRZ1 expression regulated at the transcriptional level?

The transcriptional regulation of PTPRZ1 involves several important mechanisms:

• Hypoxia-inducible factors: PTPRZ1 expression is upregulated by HIF-2α, but not HIF-1α

• Hypoxia response elements (HREs): The PTPRZ1 promoter contains multiple HREs, with HRE4 (-130 to -138) identified as the main contributor to HIF-2α-mediated upregulation

• Transcription factor cooperation: ELK1 (an Ets family transcription factor) works in concert with HIF-2α to facilitate PTPRZ1 transcription

• Promoter structure: Analysis of the PTPRZ1 promoter has identified several potential regulatory regions:

  • HRE1 (-973 to -968)

  • HRE2 (-447 to -452)

  • HRE3 (-294 to -299)

  • HRE4 (-130 to -138)

  • HRE5 (-25 to -30)

Understanding these regulatory mechanisms can inform experimental approaches to modulate PTPRZ1 expression in research contexts.

What signaling pathways are affected by PTPRZ1 activity and how can researchers monitor these effects?

PTPRZ1 is involved in several signaling pathways, with particular emphasis on:

• PTN-PTPRZ1 paracrine signaling axis: Pleiotrophin (PTN) is a ligand for PTPRZ1 that activates downstream signaling

• ERK signaling: PTN treatment significantly increases ERK phosphorylation downstream of PTPRZ1 in glioma stem cells

• Cell migration and invasion pathways: PTPRZ1 activation enhances glioma stem cell self-renewal, migration, and invasion capabilities

To monitor these signaling events, researchers can:

• Use phospho-specific antibodies to detect activated ERK (p-ERK) via Western blotting

• Employ immunoprecipitation to assess PTPRZ1 interactions with binding partners

• Utilize phosphatase activity assays to measure PTPRZ1 enzymatic function

• Analyze downstream gene expression changes using qPCR or RNA-seq

• Perform functional assays (migration, invasion, self-renewal) with and without pathway inhibitors to establish causality

How does the PTN-PTPRZ1 signaling axis contribute to glioma stem cell function?

The PTN-PTPRZ1 signaling axis plays a crucial role in regulating glioma stem cell properties:

• Self-renewal: Treatment with human recombinant PTN significantly enhances the self-renewal capacity of glioma stem cells through PTPRZ1 activation

• Migration: PTN-PTPRZ1 signaling promotes glioma stem cell migration

• Invasion: Activation of this signaling axis enhances the invasive properties of glioma stem cells

• Molecular mechanism: PTN binding to PTPRZ1 leads to increased ERK phosphorylation, which mediates these functional effects

• Therapeutic targeting: Blocking this axis with anti-PTPRZ1 antibodies (such as clone 2F10) significantly reduces these aggressive properties of glioma stem cells

This signaling axis represents a promising therapeutic target for glioblastoma, as disruption of PTN-PTPRZ1 interaction could potentially reduce tumor growth and invasion.

How can CRISPR-Cas9 technology be applied to study PTPRZ1 function in cancer models?

CRISPR-Cas9 technology offers powerful approaches for investigating PTPRZ1 function:

• Gene knockout: Design sgRNAs targeting PTPRZ1 exons to create complete loss-of-function models

• Domain-specific mutations: Introduce precise mutations to study the role of specific domains (e.g., phosphatase domain) in PTPRZ1 function

• Promoter editing: Target the HREs or other regulatory elements in the PTPRZ1 promoter to study transcriptional regulation

• Knock-in models: Create reporter constructs (e.g., fluorescent proteins) to monitor PTPRZ1 expression in real-time

• Inducible systems: Develop Tet-on/off CRISPR systems for temporal control of PTPRZ1 knockout or expression

Implementation protocol:

• Design multiple sgRNAs targeting PTPRZ1

• Package into lentiviral vectors for efficient delivery

• Transduce target cells and select for transduced cells

• Validate knockout efficiency at protein level using validated PTPRZ1 antibodies

• Perform functional assays to assess phenotypic consequences

What approaches can be used to develop and validate PTPRZ1-targeting therapeutic antibodies?

Development and validation of therapeutic anti-PTPRZ1 antibodies involves several key steps:

• Immunization and antibody generation:

  • Use recombinant PTPRZ1 extracellular domain (e.g., amino acids 26-300) as the immunizing antigen

  • Immunize suitable host animals (e.g., BALB/c mice) with a proper immunization schedule

  • Perform B cell fusion and hybridoma selection to isolate monoclonal antibodies

  • Screen for high-affinity clones using specialized affinity assays

• Validation of antibody specificity:

  • Western blotting against wildtype and PTPRZ1-knockout cells

  • Immunoprecipitation followed by mass spectrometry

  • Immunohistochemistry on tissues with known PTPRZ1 expression patterns

  • Flow cytometry on cells with differential PTPRZ1 expression

• Functional characterization:

  • Test ability to block PTN-induced ERK phosphorylation

  • Assess effects on glioma stem cell self-renewal, migration, and invasion

  • Evaluate potential cytotoxic or growth-inhibitory effects

• In vivo validation:

  • Pharmacokinetic and biodistribution studies

  • Efficacy testing in relevant tumor models

  • Safety assessment in appropriate animal models

How can researchers develop CAR T-cell therapy targeting PTPRZ1 for glioblastoma treatment?

Development of PTPRZ1-targeting CAR T-cell therapy involves several critical steps:

What are common challenges in PTPRZ1 detection by Western blotting and how can they be addressed?

Detecting PTPRZ1 by Western blotting can present several challenges:

• High molecular weight detection issues:

  • PTPRZ1 has a calculated molecular weight of 255 kDa but is typically observed at 163-175 kDa

  • Use low percentage (6-8%) gels for better resolution of high molecular weight proteins

  • Extend transfer time or use specialized transfer systems for large proteins

  • Consider pulsed-field gel electrophoresis for very large isoforms

• Multiple isoform detection:

  • PTPRZ1 has isoforms of 254 kDa, 163 kDa, and 90 kDa

  • Use gradient gels to resolve multiple isoforms in a single run

  • Confirm identities using isoform-specific antibodies or knockout controls

• Low expression levels:

  • Increase sample concentration or loading volume

  • Use enhanced chemiluminescence substrates for higher sensitivity

  • Consider signal amplification systems for very low abundance

• Non-specific binding:

  • Optimize blocking conditions (try BSA instead of milk if background persists)

  • Increase washing stringency with higher salt or detergent concentrations

  • Test different antibody dilutions (start with recommended 1:500-1:1000 range)

How should researchers validate the specificity of PTPRZ1 antibodies in their experimental systems?

Thorough validation of PTPRZ1 antibodies should include:

• Genetic controls:

  • Test antibody on PTPRZ1 knockout or knockdown samples created using sgRNA-PTPRZ1 lentivirus

  • Verify loss of signal correlates with reduced PTPRZ1 mRNA levels by RT-qPCR

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide to block specific binding

  • Compare signal with and without peptide competition

• Multiple antibody validation:

  • Use antibodies from different sources or raised against different epitopes

  • Confirm consistent detection patterns across antibodies

• Multiple techniques:

  • Compare results from Western blotting, immunohistochemistry, and immunofluorescence

  • Validate with additional techniques like mass spectrometry or immunoprecipitation

• Positive and negative controls:

  • Include known positive samples (e.g., HepG2 cells, HeLa cells, rat cerebellum)

  • Test in tissues/cells known to lack PTPRZ1 expression

What is the optimal approach for analyzing PTPRZ1 expression in tissue microarrays of brain tumors?

For analyzing PTPRZ1 expression in brain tumor tissue microarrays:

• Antibody optimization:

  • Test multiple dilutions of PTPRZ1 antibody (starting with 1:50-1:500 range)

  • Determine optimal antigen retrieval method (TE buffer pH 9.0 recommended; alternatively, citrate buffer pH 6.0)

  • Include positive control tissues (rat cerebellum) on each slide

• Staining protocol:

  • Use automated staining platforms if available for consistency across samples

  • Include chromogen development time standardization

  • Consider multiplex staining to co-localize with cell type-specific markers

• Scoring methodology:

  • Develop a standardized scoring system (e.g., H-score, combining intensity and percentage)

  • Implement digital image analysis for objective quantification

  • Ensure blinded assessment by multiple observers

• Validation steps:

  • Correlate IHC findings with mRNA expression data where available

  • Confirm expression patterns in whole tissue sections from select cases

  • Validate findings in independent cohorts

• Data analysis:

  • Correlate PTPRZ1 expression with clinicopathological parameters

  • Perform survival analysis to assess prognostic significance

  • Consider multivariate analysis to identify independent prognostic factors