PARK7 Antibody

What is PARK7/DJ-1 and what is its relevance in scientific research?

PARK7/DJ-1 is a multifunctional protein with important antioxidant properties that plays a critical protective role against oxidative stress and cell death. This protein has gained significant attention in neurodegenerative disease research, particularly in Parkinson's disease, where mutations in the PARK7 gene are linked to early-onset forms of the condition . The human PARK7 protein spans from Ala2 to Asp189, comprising 189 amino acids with a calculated molecular weight of approximately 20 kDa, though it's typically observed at 22-25 kDa in Western blot analyses due to post-translational modifications .

At the cellular level, PARK7/DJ-1 demonstrates localization in both cytoplasmic and nuclear compartments, as evidenced by immunofluorescence studies in various cell lines including HeLa cells . Beyond neurodegenerative diseases, emerging research has identified PARK7/DJ-1 as an important regulator in inflammatory pathways. Studies show that PARK7 expression is elevated in the mucosa of children with Crohn's disease compared to healthy controls, indicating its involvement in inflammatory conditions beyond neurological contexts .

For researchers investigating cellular stress responses, it's important to note that PARK7/DJ-1 expression and function can be significantly altered by different experimental conditions. Studies have demonstrated that H₂O₂ and IL-17 treatment increase PARK7 synthesis in cellular models, while LPS, TNF-α, and TGF-β treatments decrease its expression .

What antibody types are available for PARK7/DJ-1 detection and what are their specific applications?

Multiple antibody types have been developed for PARK7/DJ-1 detection, each optimized for specific applications in research settings. Understanding their characteristics is essential for selecting the appropriate tool for different experimental approaches.

Monoclonal antibodies, such as the Mouse Anti-Human/Mouse/Rat Park7/DJ-1 Monoclonal Antibody (Clone #925805R), demonstrate high specificity and are particularly effective in Western blot applications. These antibodies typically detect PARK7/DJ-1 at approximately 22 kDa under reducing conditions and have been validated in various cell lines including HeLa, MEF, and C6 rat glioma cells, demonstrating cross-species reactivity .

Recombinant monoclonal antibodies, such as the Rabbit recombinant monoclonal antibody (Clone #007), offer improved consistency between batches and can be used across multiple applications including Flow Cytometry, Immunocytochemistry/Immunofluorescence, and Western Blot .

The following table summarizes optimal dilutions for various applications based on antibody type:

For immunofluorescence applications, these antibodies reveal that PARK7/DJ-1 is primarily localized to the cytoplasm and nuclei of cells, as demonstrated in HeLa cells . When designing experiments, it's critical to validate antibody dilutions for specific experimental systems, as optimal conditions may vary based on sample type and detection method.

How should samples be prepared for optimal PARK7/DJ-1 detection?

Effective sample preparation is crucial for reliable PARK7/DJ-1 detection across different experimental platforms. Based on validated protocols in the literature, specific methodological approaches are recommended depending on the application and sample type.

For cell lysate preparation, successful PARK7/DJ-1 detection has been achieved in various cell lines including HeLa, HEK-293, BxPC-3, Jurkat, HSC-T6, MEF, and C6 cells . When preparing lysates for Western blot analysis, reducing conditions and appropriate buffer systems are essential. Specifically, researchers have reported successful detection using Immunoblot Buffer Group 1 with PVDF membranes .

For tissue samples, PARK7/DJ-1 has been successfully detected in mouse and rat brain tissues . These samples typically require careful homogenization to maintain protein integrity while ensuring adequate extraction. Mechanical disruption in the presence of protease inhibitors is generally recommended to preserve PARK7/DJ-1 epitopes and prevent degradation during the extraction process.

When preparing samples for immunofluorescence studies, immersion fixation has proven effective for preserving PARK7/DJ-1 epitopes. In validated protocols, researchers have successfully detected PARK7/DJ-1 in HeLa cells by using an immersion fixation method followed by antibody incubation at 10 μg/mL for 3 hours at room temperature .

For flow cytometry applications involving intracellular staining of PARK7/DJ-1, a recommended concentration is 0.25 μg per 10^6 cells in a 100 μl suspension . This requires appropriate permeabilization protocols to allow antibody access to the intracellular PARK7/DJ-1 protein.

Samples containing PARK7/DJ-1 should be handled with attention to protein stability. Storage buffers typically include PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For long-term storage, samples should be maintained at -20°C, with aliquoting recommended to avoid freeze-thaw cycles that can degrade the PARK7/DJ-1 protein .

How does PARK7/DJ-1 expression change under different oxidative stress conditions?

PARK7/DJ-1 expression and function demonstrate significant responsiveness to oxidative stress conditions, requiring careful methodological considerations when designing experiments to investigate these changes. Current research indicates distinct expression patterns under different stress inducers.

Hydrogen peroxide (H₂O₂) treatment has been shown to increase PARK7/DJ-1 synthesis in cellular models, suggesting an adaptive response to oxidative challenge . When designing H₂O₂ treatment protocols, researchers should implement dose-response studies and time-course analyses to capture both acute and adaptive responses. Critical controls should include measurement of cellular viability to distinguish expression changes from non-specific stress responses.

Interestingly, inflammatory mediators show divergent effects on PARK7/DJ-1 expression. IL-17 treatment increases PARK7 synthesis, while LPS, TNF-α, and TGF-β treatments decrease its expression . This differential regulation suggests complex pathway interactions that should be carefully distinguished in experimental designs. When studying these effects, time-dependent sampling is crucial, as early and late responses may differ significantly.

For in vivo oxidative stress studies, the compound 3,4,5-trimethoxy-N-(4-(8-methylimidazo(1,2-a)pyridine-2-yl)phenyl)benzamide (Comp23), which increases PARK7 activity, has been used in mouse models of DSS-colitis . This approach allows for investigation of PARK7/DJ-1's protective functions under inflammatory stress conditions.

Methodologically, researchers should consider the following technical aspects when studying oxidative stress-induced changes in PARK7/DJ-1:

• Post-translational modifications: Oxidative stress can induce oxidation of PARK7/DJ-1 at critical cysteine residues, potentially altering its function.

• Subcellular redistribution: Under stress conditions, PARK7/DJ-1 may undergo translocation between cellular compartments. Fractionation studies can capture these dynamic changes.

• Protein-protein interactions: Stress conditions may alter PARK7/DJ-1's interaction network. Co-immunoprecipitation under varying stress conditions can reveal context-dependent protein partnerships.

What methodological approaches can be used to investigate PARK7/DJ-1's role in inflammatory signaling pathways?

Investigating PARK7/DJ-1's role in inflammatory signaling pathways requires integrated approaches spanning molecular, cellular, and in vivo methodologies. Based on current research, several strategic approaches can effectively elucidate its functions in inflammatory contexts.

Gene silencing approaches provide a powerful tool for understanding PARK7/DJ-1's direct influence on inflammatory signaling. Research has demonstrated that PARK7 gene silencing influences the synthesis of key inflammatory cytokines including IL1B, IL6, TNFA, and TGFB1 . When implementing gene silencing, researchers should consider both transient (siRNA) and stable (shRNA, CRISPR-Cas9) approaches depending on the temporal aspects of the inflammatory response being studied.

Cytokine analysis methods are central to characterizing PARK7/DJ-1's impact on inflammatory responses. Multiplex assays that can simultaneously measure multiple cytokines provide comprehensive profiles of inflammatory mediators affected by PARK7/DJ-1 modulation. When designing these experiments, time-course analyses are particularly informative, as they can distinguish between early and late inflammatory responses.

The following experimental workflow has been validated for studying PARK7/DJ-1's inflammatory functions:

• Compare baseline inflammatory marker expression between wild-type and PARK7/DJ-1-deficient cells/tissues using qPCR and ELISA for key cytokines (IL-1β, IL-6, TNF-α, TGF-β1)

• Challenge both systems with inflammatory stimuli (LPS, H₂O₂, IL-17) and assess differential responses at multiple time points (typically 2, 6, 12, and 24 hours)

• Measure both transcriptional (mRNA) and translational (protein) responses to identify potential post-transcriptional regulatory mechanisms

For in vivo inflammation models, DSS-colitis models treated with Comp23 (a compound that increases PARK7 activity) have demonstrated PARK7/DJ-1's immunomodulatory effects , providing a template for similar approaches in other inflammatory models including neuroinflammatory conditions.

How can researchers effectively study PARK7/DJ-1's interaction with mitochondrial proteins?

Studying PARK7/DJ-1's interactions with mitochondrial proteins requires specialized approaches that address both the technical challenges of mitochondrial protein analysis and the biological complexity of PARK7/DJ-1's functions. Several methodological strategies are particularly effective for these investigations.

Subcellular fractionation represents a fundamental technique for isolating mitochondria to study PARK7/DJ-1 associations. For effective mitochondrial isolation, differential centrifugation protocols should be optimized to maintain the integrity of protein-protein interactions. Western blot verification of fractionation purity is essential, using markers such as VDAC or COX IV for mitochondria, GAPDH for cytosol, and nuclear markers to confirm separation efficiency.

Co-immunoprecipitation (Co-IP) studies can reveal direct interactions between PARK7/DJ-1 and mitochondrial proteins. When performing Co-IP for mitochondrial interactions, detergent selection is critical - mild detergents often preserve physiologically relevant interactions better than stronger detergents. Based on validated protocols, approximately 0.5-4.0 μg of PARK7/DJ-1 antibody is typically required for immunoprecipitation from 1.0-3.0 mg of total protein lysate .

Immunofluorescence microscopy provides valuable spatial information about PARK7/DJ-1's co-localization with mitochondrial structures. PARK7/DJ-1 has been successfully visualized in both cytoplasmic and nuclear compartments using antibodies at concentrations of 10-15 μg/mL . For co-localization studies, pairing PARK7/DJ-1 antibodies with established mitochondrial markers allows for assessment of potential associations under different experimental conditions.

For functional studies of these interactions, PARK7/DJ-1 gene silencing can help establish the functional consequences of PARK7/DJ-1 loss on mitochondrial protein expression and function. When employing gene silencing approaches, validation of knockdown efficiency by both Western blot and qPCR is recommended to ensure complete assessment of the intervention.

How can researchers validate PARK7/DJ-1 antibody specificity in their experimental systems?

Validating PARK7/DJ-1 antibody specificity is essential for ensuring experimental rigor and reproducibility. Several approaches can be implemented to confirm antibody specificity and optimize detection conditions.

Western blot analysis using PARK7/DJ-1 knockout cell lines compared to their wild-type counterparts provides the gold standard for antibody validation. This approach can identify potential cross-reactivity or non-specific binding that might otherwise confound experimental results . When performing Western blots, PARK7/DJ-1 is typically detected at approximately 22-25 kDa under reducing conditions .

Multiple antibody validation is recommended, particularly when examining novel functions or contexts for PARK7/DJ-1. Using antibodies targeting different epitopes of PARK7/DJ-1 can provide independent confirmation of observed patterns. The literature reports successful detection using both N-terminal and C-terminal targeting antibodies, which can serve as complementary validation tools.

Peptide competition assays offer another validation approach, particularly for polyclonal antibodies. Pre-incubation of the antibody with purified PARK7/DJ-1 protein or immunizing peptide should abolish or significantly reduce specific staining in Western blot, immunofluorescence, or other applications.

Cross-species validation can provide additional confidence in antibody specificity. Current antibodies have demonstrated reactivity with human, mouse, and rat PARK7/DJ-1 , allowing for comparative studies across these species. When transitioning between species, it's advisable to verify detection patterns to confirm the expected expression profiles.

When validating by immunofluorescence, attention to subcellular localization patterns is important. PARK7/DJ-1 shows characteristic distribution in both cytoplasmic and nuclear compartments . Deviations from this expected pattern might indicate non-specific binding or technical issues with the staining protocol.

What are common technical challenges when working with PARK7/DJ-1 antibodies and how can they be addressed?

Working with PARK7/DJ-1 antibodies presents several technical challenges that researchers should anticipate and address through methodological refinements.

Variability in detection sensitivity across applications is a common challenge. While an antibody may perform well in Western blot, it might show reduced sensitivity in immunofluorescence or immunoprecipitation. To address this, application-specific optimization is essential. For Western blot applications, dilutions ranging from 1:1,000 to 1:10,000 have been validated , while immunofluorescence typically requires higher concentrations (10-15 μg/mL or dilutions of 1:20-1:500) .

Background signal can complicate PARK7/DJ-1 detection, particularly in tissues with high endogenous peroxidase activity or autofluorescence. For immunohistochemistry applications, thorough blocking procedures (using 5-10% normal serum from the secondary antibody host species) and appropriate quenching of endogenous peroxidase activity are recommended. For immunofluorescence, extended blocking periods (1-2 hours) and inclusion of 0.1-0.3% Triton X-100 in blocking buffers can improve signal-to-noise ratios.

Post-translational modifications of PARK7/DJ-1, particularly under oxidative stress conditions, can alter epitope accessibility and antibody recognition. When studying PARK7/DJ-1 in oxidative stress contexts, researchers should be aware that certain modifications might affect antibody binding. Using multiple antibodies targeting different epitopes can help mitigate this challenge.

For storage and handling, PARK7/DJ-1 antibodies are typically stable when stored at -20°C, with aliquoting recommended to avoid freeze-thaw cycles . For working solutions, storage at 4°C with preservatives such as 0.02% sodium azide can maintain antibody performance for 1-2 weeks.

When troubleshooting inconsistent results, systematic optimization of key parameters is recommended:

• Titrate antibody concentration across a broader range than manufacturer recommendations

• Test multiple antigen retrieval methods (for fixed tissues or cells)

• Vary incubation times and temperatures

• Optimize buffer compositions, particularly detergent concentrations for membrane permeabilization

How can PARK7/DJ-1 antibodies be utilized in the study of neurodegenerative diseases?

PARK7/DJ-1 antibodies offer valuable tools for investigating neurodegenerative disease mechanisms, particularly in Parkinson's disease where PARK7 mutations are linked to early-onset forms of the condition . Several methodological approaches leverage these antibodies for specific neurodegenerative disease applications.

Immunohistochemistry and immunofluorescence using PARK7/DJ-1 antibodies enable spatial characterization of PARK7/DJ-1 expression patterns in neural tissues. In post-mortem brain samples or animal models, these techniques can reveal changes in PARK7/DJ-1 distribution across different brain regions affected by neurodegenerative processes. PARK7/DJ-1 antibodies have been successfully used at concentrations of 10-15 μg/mL for these applications .

For protein-protein interaction studies relevant to neurodegeneration, co-immunoprecipitation using PARK7/DJ-1 antibodies can reveal disease-relevant binding partners. The recommended protocol involves using 0.5-4.0 μg of antibody per 1-3 mg of total protein lysate . This approach can identify novel interactions that may contribute to neuroprotective functions or be disrupted in disease states.

Western blot analysis using PARK7/DJ-1 antibodies enables quantitative assessment of expression levels in different experimental models of neurodegeneration. Validated dilutions range from 1:2,000 to 1:10,000 for this application . When comparing expression across different brain regions or disease states, normalization to appropriate housekeeping proteins is essential for accurate quantification.

Flow cytometry applications using PARK7/DJ-1 antibodies (at dilutions of 1:25-1:100) can assess PARK7/DJ-1 expression in isolated neural cell populations, allowing for cell type-specific analysis. This approach is particularly valuable for examining differential expression or alterations in microglia, astrocytes, and neurons in neurodegenerative contexts.

In cellular models of neurodegeneration, PARK7/DJ-1 antibodies can track changes in expression or localization following exposure to neurotoxins, oxidative stressors, or genetic manipulations. This enables mechanistic studies connecting PARK7/DJ-1 function to neuroprotective pathways that may be therapeutic targets.

What are the emerging applications for PARK7/DJ-1 antibodies in inflammatory disease research?

Recent discoveries have expanded PARK7/DJ-1's relevance beyond neurodegenerative conditions to include inflammatory diseases, creating new applications for PARK7/DJ-1 antibodies in this research area.

Immunohistochemical analysis of inflammatory tissues using PARK7/DJ-1 antibodies has revealed elevated expression in the mucosa of children with Crohn's disease compared to healthy controls . This suggests potential applications for PARK7/DJ-1 antibodies in identifying disease-associated changes in various inflammatory conditions. When applying these techniques to inflammatory tissues, optimization of antigen retrieval methods is often necessary to overcome potential epitope masking in inflamed samples.

For mechanistic studies, PARK7/DJ-1 antibodies can be employed to monitor expression changes following exposure to inflammatory mediators. Research has demonstrated that H₂O₂ and IL-17 increase PARK7/DJ-1 synthesis, while LPS, TNF-α, and TGF-β decrease its expression . Western blot analysis (at dilutions of 1:1,000-1:10,000) can quantitatively track these changes across different inflammatory conditions and time points.

In functional studies, combining PARK7/DJ-1 antibodies with gene silencing approaches enables comprehensive analysis of PARK7/DJ-1's role in inflammatory signaling. Research has shown that PARK7 gene silencing influences the synthesis of inflammatory cytokines including IL1B, IL6, TNFA, and TGFB1 . Antibody-based detection methods can confirm knockdown efficiency and monitor subsequent changes in inflammatory markers.

Flow cytometry applications using PARK7/DJ-1 antibodies can assess expression levels across different immune cell populations, potentially revealing cell type-specific roles in inflammatory responses. This approach requires appropriate permeabilization protocols and antibody concentrations of approximately 0.25 μg per 10^6 cells .

For translational research connecting PARK7/DJ-1 to human inflammatory diseases, immunostaining of patient-derived samples with PARK7/DJ-1 antibodies can correlate expression patterns with clinical disease activity and treatment response. This approach may identify PARK7/DJ-1 as a potential biomarker or therapeutic target in inflammatory conditions.