GSTP1 Antibody

What is GSTP1 and why is it significant in scientific research?

GSTP1 is a member of the Glutathione S-transferase family that plays a crucial role in the metabolic detoxification of various environmental carcinogens and lipid hydroperoxides. This enzyme is essential for cellular protection against oxidative stress by neutralizing reactive oxygen species and harmful electrophilic compounds. GSTP1 becomes upregulated in response to oxidative stress, highlighting its importance in maintaining cellular homeostasis .

Additionally, the GSTP1 gene is subject to CpG island hypermethylation, a modification that has been associated with human prostatic carcinogenesis and other cancer types . This epigenetic modification can be detected in biological fluids such as urine, ejaculate, and plasma from men diagnosed with prostate cancer, making GSTP1 a valuable biomarker for cancer research .

What types of GSTP1 antibodies are available and how do they differ in research applications?

Based on current research resources, several types of GSTP1 antibodies are available for research purposes:

• Monoclonal antibodies:

  • GSTP1 Antibody (F-6): A mouse monoclonal IgG2a antibody that detects human GSTP1 through Western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA)

  • GSTP1 Antibody (3F2C2): A mouse monoclonal IgG1 kappa light chain antibody validated for WB, IP, IF, and immunohistochemistry with paraffin-embedded sections (IHC-P)

• Various conjugated forms:

  • Non-conjugated primary antibodies

  • Agarose-conjugated antibodies optimized for immunoprecipitation

  • HRP-conjugated antibodies for direct detection in Western blotting

  • Fluorescent-conjugated antibodies (PE, FITC, Alexa Fluor®) for flow cytometry and IF applications

The key differences between these antibodies include their isotype (IgG1 vs. IgG2a), epitope recognition, and validated applications. Selection should be based on the intended experimental methodology, target species, and specific research objectives.

What experimental validation should be performed before using GSTP1 antibodies in a new research system?

Before implementing GSTP1 antibodies in a new research system, several validation steps are essential:

• Specificity verification:

  • Test antibodies on positive control samples (cells/tissues known to express GSTP1)

  • Include negative controls (samples with known GSTP1 silencing via promoter hypermethylation)

  • Verify the expected molecular weight (~23 kDa) in Western blotting applications

• Cross-validation approaches:

  • Use multiple GSTP1 antibodies targeting different epitopes

  • Correlate protein detection with mRNA expression analysis

  • For IHC applications, validate that mRNA expression is concordant with protein staining, as demonstrated in the literature

• Application-specific validation:

  • For Western blotting: Optimize sample preparation conditions (reducing vs. non-reducing), antibody dilutions, and detection methods

  • For IHC/IF: Determine optimal antigen retrieval methods, antibody concentrations, and incubation times

  • For cancer tissue analysis: Be aware that GSTP1 silencing through promoter hypermethylation is common in certain cancers (90-95% of prostate cancers)

Thorough validation ensures reliable and reproducible results when implementing GSTP1 antibodies in new experimental systems.

What is the optimal protocol for Western blot detection of GSTP1 protein?

For optimal Western blot detection of GSTP1, researchers should follow this methodological approach:

• Sample preparation:

  • Prepare samples in LDS sample loading buffer containing reducing agent

  • Boil samples for 10 minutes to ensure complete protein denaturation

  • For phosphorylated GSTP1 detection, non-reducing conditions may be required (samples without reducing agents and without heating prior to electrophoresis)

• Electrophoresis and transfer:

  • Use 10% Bis-Tris gels in MOPS buffer for optimal separation

  • Electrophoretically transfer proteins to Immobilon P membrane (or equivalent PVDF membrane)

  • Verify transfer efficiency by staining with Coomassie Brilliant Blue G-250

• Antibody incubation:

  • Block in 1× TBS-T containing 5% BSA

  • Incubate with appropriately diluted primary GSTP1 antibody overnight at 4°C

  • Apply horseradish peroxidase-conjugated secondary antibody

  • Visualize immunoreactive bands with an ECL system

• Controls and interpretation:

  • Include loading controls (β-actin)

  • Include positive controls (cells known to express GSTP1)

  • For studies examining post-translational modifications, include appropriate controls (e.g., anti-phosphotyrosine antibodies for phosphorylation studies)

This protocol has been validated in multiple research contexts and optimizes detection sensitivity while minimizing background interference.

How can researchers effectively assess GSTP1 methylation status in experimental samples?

Methylation-specific PCR (MSP) is the established technique for assessing GSTP1 promoter methylation. Based on published methodologies, researchers should follow this approach:

• DNA extraction and bisulfite modification:

  • Extract genomic DNA from tissue biopsies or cell lines

  • Perform sodium bisulfite modification of DNA, which converts unmethylated cytosines to uracils while leaving methylated cytosines unchanged

• PCR amplification:

  • Design primers specific for methylated and unmethylated versions of the GSTP1 promoter region

  • Perform separate PCR reactions for methylated and unmethylated sequences

  • Include appropriate controls (methylated and unmethylated standards)

• Analysis:

  • Analyze PCR products on 2% agarose gel

  • Repeat at least 25% of samples to confirm reproducibility of results

  • For more quantitative assessment, consider quantitative MSP (qMSP) or pyrosequencing

• Validation:

  • Correlate methylation results with GSTP1 protein expression using immunohistochemistry or Western blotting

  • GSTP1 promoter hypermethylation is often associated with loss of protein expression

This methodology allows for reliable assessment of GSTP1 methylation status, which is particularly relevant in cancer research where GSTP1 promoter hypermethylation is a frequent event.

What are the critical parameters for immunohistochemical detection of GSTP1 in tissue samples?

For effective immunohistochemical detection of GSTP1, researchers should optimize these critical parameters:

• Tissue preparation:

  • Use formalin-fixed, paraffin-embedded (FFPE) sections at 4-6 μm thickness

  • Implement standardized fixation protocols to ensure consistent epitope preservation

• Antigen retrieval:

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Optimization of retrieval conditions may be necessary depending on fixation time and tissue type

• Antibody selection and incubation:

  • Use antibodies validated for IHC-P, such as the GSTP1 Antibody (3F2C2)

  • Optimize antibody dilution through titration experiments

  • Include appropriate controls:

    • Positive control tissues (normal tissues known to express GSTP1)

    • Negative controls (tissues with known GSTP1 silencing or omission of primary antibody)

• Detection system:

  • Select an appropriate detection system (e.g., polymer-based systems for increased sensitivity)

  • Develop signal using DAB or other suitable chromogen

  • Counterstain with hematoxylin for nuclear visualization

• Interpretation considerations:

  • Evaluate cytoplasmic and potentially nuclear staining patterns

  • Quantify staining intensity and percentage of positive cells

  • Be aware that GSTP1 expression may be lost due to promoter hypermethylation in certain cancers

  • Validation with mRNA in situ hybridization may be valuable as demonstrated in research showing concordance between GSTP1 mRNA expression and protein staining

Attention to these parameters ensures reliable and reproducible immunohistochemical detection of GSTP1 in tissue samples.

How does GSTP1 expression vary across different cancer types and what are the underlying mechanisms?

GSTP1 expression exhibits significant variations across different cancer types through several key mechanisms:

• Epigenetic silencing:

  • GSTP1 is silenced by CpG island DNA hypermethylation in 90-95% of prostate cancers

  • One-third of breast tumor biopsies (74/215) showed GSTP1 promoter hypermethylation in published studies

  • This epigenetic silencing leads to loss of GSTP1 protein expression and reduced detoxification capability

• Race-associated expression patterns:

  • GSTP1-positive prostate cancers are significantly more common in Black men compared to White men

  • Research shows that 14.9% of prostate adenocarcinomas from Black patients had GSTP1-positive tumor microarray cores, which was 2.5 times higher than the percentage from White patients (5.9%)

  • The percentage of tumors from Black patients with all tumor microarray spots positive for GSTP1 (9.5%) was 3-fold higher than the percentage from White patients (3.2%)

• Association with molecular subtypes:

  • GSTP1 positivity has been found to be enriched in ERG-positive prostate cancers among Black men

  • Estrogen (ER) and progesterone (PR) receptor-positive breast tumors showed higher frequency of GSTP1 methylation

These variations in GSTP1 expression patterns have significant implications for understanding cancer biology and developing targeted therapeutic approaches, particularly in addressing cancer disparities among different populations.

What is the relationship between GSTP1 phosphorylation and cancer cell signaling pathways?

GSTP1 phosphorylation represents a critical intersection between detoxification processes and cancer signaling pathways:

• EGFR-mediated GSTP1 phosphorylation:

  • Research has demonstrated that the Epidermal Growth Factor Receptor (EGFR) can directly phosphorylate GSTP1 on tyrosine residues

  • This phosphorylation has been examined in human brain tumor cell lines and xenografts, with functional effects observed both in vitro and in vivo

• Experimental protocols for studying GSTP1 phosphorylation:

  • Cell-free systems using recombinant GSTP1, EGFR active kinase domain, and ATP can demonstrate this phosphorylation

  • Protocols typically involve preincubating GSTP1 with GSH (5 mM) to mimic intracellular conditions

  • Analysis requires non-reducing SDS-PAGE conditions followed by Western blotting with anti-phosphotyrosine antibodies

• Connection to JNK signaling pathway:

  • Evidence suggests relationships between GSTP1, EGFR, and the JNK signaling pathway

  • Experimental setups examining this relationship include GSTP1, c-Jun, activated JNK1α1, and EGFR active catalytic domain

  • This suggests GSTP1 phosphorylation may influence stress response pathways mediated by JNK

• Implications for cancer biology:

  • GSTP1 has been described as a "caretaker gene whose loss increases cell survival in response to protracted oxidative stress"

  • The phosphorylation state of GSTP1 may influence its ability to regulate cellular stress responses and survival pathways

  • This positions GSTP1 at the intersection of detoxification processes and signaling pathways that control cell fate

This connection between GSTP1 phosphorylation and signaling pathways suggests more complex roles for GSTP1 in cancer biology beyond its classical detoxification function.

How do GSTP1 polymorphisms affect enzyme activity and cancer susceptibility?

GSTP1 polymorphisms significantly impact enzyme function and cancer susceptibility through several mechanisms:

These findings highlight the complex relationship between GSTP1 genetic variations and cancer susceptibility, involving both direct effects on enzyme activity and potential interactions with environmental and epigenetic factors.

How can researchers design experiments to investigate GSTP1's role in oxidative stress response pathways?

Designing robust experiments to investigate GSTP1's role in oxidative stress response requires multifaceted approaches:

• Cellular models and manipulation strategies:

  • Generate cellular models with varied GSTP1 expression levels:

    • GSTP1 overexpression systems (e.g., UW228₁C cells created from parental UW228 cells via stable transfection with human GSTP1 C allelic variant)

    • GSTP1 knockdown/knockout models using siRNA or CRISPR-Cas9

    • Cell lines with natural GSTP1 silencing due to promoter hypermethylation

• Oxidative stress induction protocols:

  • Implement controlled oxidative stress conditions using:

    • H₂O₂ treatment at various concentrations and time points

    • Glutathione depletion using buthionine sulfoximine

    • Exposure to environmental carcinogens or lipid hydroperoxides

  • Measure cellular responses using:

    • Cell viability assays (MTT, XTT)

    • ROS detection (DCF-DA fluorescence, EPR spectroscopy)

    • Lipid peroxidation markers (MDA, 4-HNE levels)

• Signaling pathway analysis:

  • Investigate GSTP1's interaction with JNK pathway:

    • Assess JNK activation (phospho-JNK levels)

    • Measure downstream targets (phospho-c-Jun, AP-1 activity)

    • Perform cell-free JNK kinase assays with recombinant proteins

  • Examine EGFR-GSTP1 interaction:

    • EGFR-mediated GSTP1 phosphorylation assays

    • Effects of EGFR inhibitors on GSTP1 function

    • Impact of GSTP1 phosphorylation on its detoxification activity

• Integrated multi-omics approaches:

  • Transcriptomic analysis to identify GSTP1-dependent gene expression changes under oxidative stress

  • Proteomic studies to detect GSTP1 interaction partners in stress response pathways

  • Metabolomic analysis focusing on glutathione metabolism and detoxification products

These comprehensive experimental approaches will enable researchers to elucidate GSTP1's multifaceted roles in oxidative stress responses beyond its classical detoxification function.

What methodological approaches can identify novel GSTP1 post-translational modifications?

To identify novel GSTP1 post-translational modifications (PTMs), researchers should implement these advanced methodological approaches:

• Mass spectrometry-based PTM profiling:

  • Immunoprecipitate GSTP1 using validated antibodies such as GSTP1 Antibody (F-6) or (3F2C2)

  • Perform tryptic digestion followed by LC-MS/MS analysis

  • Implement database searches with variable modifications including phosphorylation, acetylation, ubiquitination, and SUMOylation

  • Use targeted MS approaches (PRM/MRM) for quantitative analysis of specific modifications

• Phosphorylation-specific analytical techniques:

  • Expand on established EGFR-mediated GSTP1 phosphorylation protocols :

    • Use recombinant GSTP1 preincubated with 5 mM GSH

    • Incubate with various kinases (beyond EGFR) in kinase buffer with ATP

    • Analyze by non-reducing SDS-PAGE and Western blotting with phospho-specific antibodies

  • Implement phospho-proteomics approaches:

    • Phospho-enrichment techniques (IMAC, TiO₂)

    • Phospho-specific antibodies against common motifs

• Stress-induced modification analysis:

  • Expose cells to various stressors (oxidative stress, electrophiles, chemotherapeutics)

  • Compare GSTP1 modification profiles before and after stress induction

  • Correlate modifications with changes in GSTP1 activity, localization, or protein interactions

• Site-directed mutagenesis validation:

  • Generate GSTP1 mutants at putative modification sites

  • Express wild-type and mutant forms in cellular models

  • Compare functional consequences of mutations on:

    • Catalytic activity

    • Protein stability

    • Protein-protein interactions (particularly with JNK and EGFR)

    • Cellular stress responses

These methodological approaches will enable identification of novel GSTP1 PTMs and their functional significance in cellular physiology and pathological conditions, potentially revealing new therapeutic targets and biomarkers.

How can researchers examine GSTP1's role at the intersection of detoxification and cell signaling pathways?

To examine GSTP1's dual role in detoxification and cell signaling, researchers should implement these sophisticated experimental approaches:

• Protein interaction network analysis:

  • Perform comprehensive immunoprecipitation studies:

    • Use GSTP1 antibodies with agarose conjugates for efficient pulldown

    • Analyze interacting partners by mass spectrometry

    • Validate key interactions by co-immunoprecipitation and proximity ligation assays

  • Map interactions under various conditions:

    • Basal vs. stress conditions

    • Wild-type vs. mutant GSTP1

    • Phosphorylated vs. non-phosphorylated GSTP1

• Functional investigation of GSTP1-JNK interaction:

  • Implement cell-free JNK kinase assays as described in the literature:

    • Combine recombinant GSTP1 (preincubated with GSH), c-Jun, JNK1α1, and EGFR catalytic domain

    • Analyze effects on c-Jun phosphorylation by Western blotting

  • Extend to cellular systems:

    • Compare JNK pathway activation in GSTP1-positive vs. GSTP1-negative cells

    • Assess impact of GSTP1 phosphorylation state on JNK inhibition

    • Examine consequences for apoptosis and stress responses

• GSTP1 role in EGFR signaling modulation:

  • Investigate bidirectional relationship between GSTP1 and EGFR:

    • EGFR-mediated GSTP1 phosphorylation

    • Potential GSTP1 effects on EGFR signaling outputs

  • Examine functional consequences in relevant cancer models:

    • Glioblastoma cell lines (U87MG.wtEGFR mentioned in research)

    • Patient-derived xenografts (GBM6 and GBM10)

• Integration of detoxification and signaling functions:

  • Design experiments to test how GSTP1's detoxification capacity affects its signaling roles:

    • Enzyme activity assays with various substrates

    • GSH depletion/repletion studies

    • Structure-function analyses using GSTP1 mutants with altered catalytic properties

  • Examine how signaling events modify GSTP1's detoxification function:

    • Effect of phosphorylation on enzyme kinetics

    • Changes in substrate specificity

    • Alterations in protein stability or cellular localization

These integrated approaches will help elucidate GSTP1's complex functions at the intersection of detoxification processes and signaling pathways, potentially revealing novel therapeutic strategies targeting GSTP1 in various disease contexts.

What are the critical considerations for studying racial differences in GSTP1 expression patterns?

Research has revealed significant racial differences in GSTP1 expression patterns, particularly in prostate cancer. When investigating these differences, researchers should consider several critical methodological factors:

• Sample collection and representation:

  • Ensure adequate representation of diverse racial and ethnic groups

  • Published research found that 14.9% of prostate adenocarcinomas from Black patients had GSTP1-positive TMA cores compared to only 5.9% from White patients

  • Sample sizes must be sufficient to detect statistically significant differences (the cited study examined 1673 primary prostatic adenocarcinomas)

• Multidimensional analysis approach:

  • Integrate multiple analytical methods:

    • Immunohistochemistry for protein expression

    • In situ hybridization for mRNA expression (used to validate that "GSTP1 mRNA expression was concordant with protein staining")

    • Methylation analysis of the GSTP1 promoter

    • Genotyping for GSTP1 polymorphisms

• Control for confounding variables:

  • Account for potentially confounding factors:

    • Age, socioeconomic status, and access to healthcare

    • Tumor grade, stage, and molecular subtypes

    • Treatment history and comorbidities

  • Statistical approaches should include multivariate analyses

• Molecular context consideration:

  • Examine associations with other molecular alterations (the study found "GSTP1 positivity was enriched in ERG positive cancers among Black men")

  • Consider combined molecular signatures rather than isolated biomarkers

  • Investigate potential mechanisms underlying observed differences

• Biological and clinical implications:

  • Connect molecular findings to biological hypotheses

  • Published research suggested that GSTP1-positive cases may "represent a distinct molecular subtype of prostate cancer" that could provide "a biological underpinning for the observed disparate outcomes for Black men"

  • Explore therapeutic and prognostic implications of observed differences

These methodological considerations ensure robust and clinically relevant investigations of racial differences in GSTP1 expression patterns, potentially contributing to addressing cancer disparities.

How can researchers troubleshoot inconsistent GSTP1 detection in cancer samples?

When encountering inconsistent GSTP1 detection in cancer samples, researchers should implement these troubleshooting approaches:

• Address biological variability factors:

  • Heterogeneous GSTP1 promoter methylation:

    • One-third of breast tumors show GSTP1 promoter hypermethylation

    • In prostate cancer, 90-95% show GSTP1 silencing through hypermethylation

    • Perform parallel methylation analysis using methods like MSP to correlate with protein expression

  • Intratumoral heterogeneity:

    • Research has shown that some cases have GSTP1 positivity "in at least one TMA core" (7.7%) while fewer have positivity "in all TMA cores" (4.4%)

    • Use multiple cores or sections from different tumor regions

    • Consider whole section analysis rather than TMA for heterogeneous tumors

• Optimize technical parameters:

  • Antibody selection and validation:

    • Compare different antibody clones (F-6 vs. 3F2C2)

    • Validate antibody performance on known positive and negative controls

  • Antigen retrieval optimization:

    • Test multiple antigen retrieval methods (citrate vs. EDTA buffers)

    • Optimize heating time and temperature

  • Detection system sensitivity:

    • Implement signal amplification methods for low-expressing samples

    • Consider more sensitive detection systems (polymer-based vs. ABC method)

• Implement multi-modal confirmation:

  • Correlate protein with mRNA expression:

    • Use in situ hybridization to confirm GSTP1 mRNA expression

    • Consider RT-PCR or RNA-seq for quantitative assessment

  • Integrate with methylation analysis:

    • Correlate protein expression with promoter methylation status

    • Understand that hypermethylation is often associated with loss of expression

• Controls and standardization:

  • Include consistent positive and negative controls in each experiment

  • Implement standardized scoring systems for immunohistochemistry

  • Document all technical parameters to ensure reproducibility

These troubleshooting approaches address both biological and technical factors contributing to inconsistent GSTP1 detection, leading to more reliable and interpretable results in cancer research.

What are best practices for integrating GSTP1 genotype, expression, and methylation data in comprehensive studies?

Integrating GSTP1 genotype, expression, and methylation data requires robust methodological approaches and analytical frameworks:

• Unified sample processing strategy:

  • Implement DNA/RNA/protein co-extraction protocols from the same specimen

  • Document all preservation methods and extraction conditions

  • Maintain consistent sample identifiers across all analyses

• Standardized methodological approaches:

  • Genotyping: Use PCR-RFLP for Ile105Val polymorphism detection

    • Amplify DNA using specific GSTP1 primers

    • Digest PCR products with BsmAI restriction enzyme

    • Analyze digested products on 2% agarose gel

  • Methylation analysis: Implement methylation-specific PCR

    • Perform sodium bisulfite modification of genomic DNA

    • Use primers specific for methylated and unmethylated sequences

    • Consider quantitative methods for precise methylation assessment

  • Expression analysis:

    • Protein level: Use validated antibodies for Western blotting and IHC

    • mRNA level: Implement in situ hybridization or qRT-PCR

    • Correlate protein with mRNA expression for validation

• Data integration framework:

  • Implement consistent quantification methods:

    • For IHC: Standardized scoring of staining intensity and percentage

    • For methylation: Binary (methylated/unmethylated) or quantitative (% methylation)

    • For genotyping: Categorize as homozygous wild-type, heterozygous, or homozygous variant

  • Statistical integration approaches:

    • Correlation analyses between methylation status and expression

    • Association tests between genotype and methylation/expression

    • Multivariate models incorporating all molecular data with clinical parameters

• Validation strategies:

  • Internal validation through technical replicates

    • "Repeat at least 25% of the samples to confirm the results" as recommended in the literature

  • Cross-platform validation

    • Verify methylation effects on expression using multiple techniques

    • Confirm genotype-phenotype associations across different experimental systems

• Data interpretation considerations:

  • Consider context-dependent effects:

    • Research indicates that "Estrogen (ER) and progesterone (PR) receptor-positive tumors had a higher methylation frequency"

    • GSTP1 positivity patterns differ significantly between racial groups

  • Account for potential independence of mechanisms:

    • Studies indicate "Genotype and tumor methylation associations were not observed"

    • This suggests these may be independent mechanisms affecting GSTP1 function

These integrated approaches enable comprehensive characterization of GSTP1 alterations across genetic, epigenetic, and protein levels, providing deeper insights into GSTP1 biology in health and disease contexts.