PARK7 P1E12AT Antibody

What is the PARK7 P1E12AT Antibody and what is its target protein?

The PARK7 P1E12AT Antibody is a Mouse Anti-Human Monoclonal antibody that specifically targets PARK7/DJ-1, a multifunctional protein involved in various cellular processes including oxidative stress response, gene transcription regulation, and neuroprotection. This antibody is derived from hybridization of mouse F0 myeloma cells with spleen cells from BALB/c mice immunized with recombinant human PARK7 protein (amino acids 1-189) purified from E. coli expression systems . The target protein, PARK7/DJ-1, has significant relevance in Parkinson's disease research, as mutations in this gene are associated with autosomal recessive early-onset Parkinson's disease (PARK7) .

What are the optimal storage conditions for maintaining antibody activity?

For short-term storage (up to 1 month), the antibody should be stored at 4°C. For longer-term storage, -20°C is recommended to maintain stability and activity. Critically, researchers must avoid freeze-thaw cycles as these can significantly degrade antibody performance . The antibody is typically provided in a formulation of 1mg/ml containing PBS (pH 7.4), 10% Glycerol, and 0.02% Sodium Azide, which helps maintain stability . The expected shelf life is 12 months at -20°C when properly stored.

What are the validated applications for PARK7 P1E12AT Antibody and optimal working dilutions?

The PARK7 P1E12AT Antibody has been validated for several experimental applications:

Researchers should note that optimal dilutions should be determined empirically for each experimental system and application .

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

For Western blot analysis, cells should be lysed in a buffer containing appropriate protease inhibitors to prevent degradation of the target protein. For neuronal samples specifically, it is recommended to include both protease and phosphatase inhibitors due to the extensive post-translational modification network in neuronal tissues . When studying PARK7 secretion, cells should be cultured in serum-free medium to prevent contamination by serum proteins that might interfere with analysis . For subcellular localization studies, it's important to note that PARK7/DJ-1 is predominantly found in the cytosolic protein-enriched fraction (approximately 80%) with smaller amounts in other cellular compartments .

What controls should be included when studying PARK7/DJ-1 secretion?

When investigating PARK7/DJ-1 secretion, several critical controls are necessary:

• FN1 (fibronectin 1) should be used as a protein marker secreted via the conventional pathway

• RPN1 (ribophorin I) should be used as a cell-resident protein control

• LDH (lactate dehydrogenase) release should be measured to verify that protein detection in the medium is not due to plasma membrane leakage or cell death

• Brefeldin A treatment can be used to distinguish between conventional ER/Golgi-dependent secretion and unconventional secretory pathways

These controls are essential for validating that observed PARK7/DJ-1 in the extracellular space is due to active secretion rather than cellular damage or experimental artifacts.

How can researchers distinguish between oxidized and non-oxidized forms of PARK7/DJ-1?

PARK7/DJ-1 oxidation state is a critical functional parameter, particularly in oxidative stress research. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the recommended method for distinguishing between oxidized and non-oxidized forms of PARK7/DJ-1. This technique separates proteins based on both isoelectric point and molecular weight, allowing visualization of charge shifts caused by oxidation of specific residues, particularly Cys106 . Researchers should note that the ratio of oxidized PARK7 to total PARK7 in medium is typically similar to that observed in cells, suggesting that secretion is not selectively induced by oxidation . For accurate quantification, densitometric analysis of the ratio between oxidized and non-oxidized forms should be performed.

What are common pitfalls in PARK7/DJ-1 antibody-based detection and how can they be avoided?

Several technical issues can complicate PARK7/DJ-1 detection:

• Cross-reactivity: Due to evolutionary conservation of PARK7/DJ-1, antibodies may cross-react with homologs from different species. The P1E12AT clone has been specifically developed against human PARK7 but shows cross-reactivity with mouse PARK7/DJ-1 .

• Multiple bands: Detection of multiple bands (20 kDa, 25 kDa, and sometimes 30 kDa) can confuse data interpretation. These represent different post-translationally modified forms of PARK7/DJ-1 .

• Background signal: High background can be reduced by optimizing blocking conditions (5% non-fat milk or BSA) and increasing the number or duration of washing steps.

• Subcellular fractionation artifacts: When studying localization, gentle lysis techniques should be employed to prevent redistribution of PARK7/DJ-1 between cellular compartments.

• Antibody batch variability: Validation should be performed for each new lot using positive control samples (HeLa, MCF-7, or SH-SY5Y cell lysates) .

How can researchers validate PARK7/DJ-1 antibody specificity?

To ensure antibody specificity, researchers should:

• Include PARK7/DJ-1 knockout or knockdown samples as negative controls

• Perform peptide competition assays by pre-incubating the antibody with excess recombinant PARK7/DJ-1 protein

• Compare detection patterns across multiple antibodies targeting different epitopes of PARK7/DJ-1

• Confirm results with mass spectrometry when possible

• Verify that detection is lost in PARK7-deficient cell lines or tissues

Several publications have utilized PARK7/DJ-1 knockout models for antibody validation, providing an excellent resource for positive and negative control data .

How can PARK7 P1E12AT Antibody be utilized to study unconventional secretion mechanisms?

PARK7/DJ-1 secretion occurs via an unconventional secretory pathway, making it a valuable model for studying this process. Unlike proteins secreted through the conventional ER/Golgi pathway, PARK7/DJ-1 secretion is not inhibited by brefeldin A . To study this mechanism:

• Treat cells with 6-hydroxydopamine (6-OHDA), which enhances PARK7/DJ-1 secretion through the unconventional pathway

• Compare secretion in the presence/absence of brefeldin A to confirm the unconventional pathway

• Analyze PARK7/DJ-1 in subcellular fractions to track its movement

• Examine the effects of calcium signaling modulators, as unconventional secretion is often calcium-dependent

• Use live-cell imaging with fluorescently-tagged PARK7/DJ-1 to visualize secretion events in real-time

This approach allows researchers to elucidate the specific mechanisms governing PARK7/DJ-1 secretion, which may provide insights into both Parkinson's disease pathology and fundamental cellular trafficking processes.

What methodological approaches can be used to study the neuroprotective function of PARK7/DJ-1?

To investigate PARK7/DJ-1's neuroprotective role:

• Oxidative stress models: Expose neuronal cells (SH-SY5Y, primary neurons) to hydrogen peroxide or 6-OHDA with varying levels of PARK7/DJ-1 expression (overexpression, knockdown, or knockout)

• Mitochondrial function assessment: Measure mitochondrial membrane potential, oxygen consumption rate, and ATP production in relation to PARK7/DJ-1 levels

• Protein-protein interaction studies: Use the P1E12AT antibody for co-immunoprecipitation to identify PARK7/DJ-1 binding partners under normal and stress conditions

• Post-translational modification analysis: Examine how oxidative stress affects PARK7/DJ-1 modifications using the antibody in combination with phospho-specific or ubiquitin-specific antibodies

• In vivo models: Analyze PARK7/DJ-1 expression in animal models of Parkinson's disease using immunohistochemistry with the P1E12AT antibody

These approaches can help elucidate how PARK7/DJ-1 regulates mitochondrial uncoupling proteins in dopaminergic neurons and provides protection against oxidative stress-induced cell death .

How can researchers investigate the relationship between PARK7/DJ-1 mutations and Parkinson's disease?

To study PARK7/DJ-1 mutations in Parkinson's disease context:

• Mutation analysis: Generate cell lines expressing PARK7/DJ-1 variants (such as L166P, which reduces protein stability) and compare antibody detection of wild-type versus mutant proteins

• Protein stability assessment: Perform cycloheximide chase experiments to determine half-life differences between wild-type and mutant PARK7/DJ-1

• Functional assays: Compare the antioxidant capacity of wild-type versus mutant PARK7/DJ-1 by measuring ROS levels and cell viability under oxidative stress

• Subcellular localization: Use immunofluorescence with P1E12AT antibody to determine if mutations alter PARK7/DJ-1 localization patterns

• Patient-derived samples: Compare PARK7/DJ-1 expression, modification, and function in patient-derived fibroblasts or iPSC-derived neurons using the antibody

These methodologies can help establish the pathological mechanisms by which PARK7/DJ-1 mutations contribute to neurodegeneration in Parkinson's disease.

How might the PARK7 P1E12AT Antibody be used in biomarker development for Parkinson's disease?

The secretory nature of PARK7/DJ-1 suggests potential applications in biomarker development:

• Develop quantitative assays (sandwich ELISA, MSD) using the P1E12AT antibody to measure PARK7/DJ-1 levels in patient biofluids (CSF, plasma, saliva)

• Compare PARK7/DJ-1 levels and oxidation state between Parkinson's disease patients and healthy controls

• Correlate PARK7/DJ-1 levels with disease progression, severity, and response to treatment

• Examine whether PARK7/DJ-1 secretion is altered by environmental stressors or genetic risk factors

• Develop multiplexed approaches to simultaneously measure PARK7/DJ-1 along with other Parkinson's disease-related proteins (α-synuclein, LRRK2)

This approach could lead to the development of novel diagnostic tools or therapeutic monitoring methods for Parkinson's disease.

What methodological considerations apply when studying PARK7/DJ-1 in different model systems?

Different experimental models require specific considerations:

Researchers should validate the antibody in their specific model system before proceeding with extensive experimentation .

How can researchers integrate PARK7/DJ-1 studies with broader neurodegeneration research?

To connect PARK7/DJ-1 research to broader neurodegenerative mechanisms:

• Investigate interactions between PARK7/DJ-1 and other Parkinson's disease-associated proteins (α-synuclein, PINK1, Parkin)

• Examine PARK7/DJ-1's role in processes common to multiple neurodegenerative diseases (protein aggregation, mitochondrial dysfunction, neuroinflammation)

• Apply the P1E12AT antibody in multi-label immunofluorescence to explore co-localization with markers of cellular stress or pathological inclusions

• Utilize the antibody in proximity ligation assays to identify novel PARK7/DJ-1 interaction partners in neural tissues

• Explore how PARK7/DJ-1 function is affected by aging, the primary risk factor for neurodegenerative diseases

This integrative approach can help position PARK7/DJ-1 research within the broader context of neurodegeneration, potentially revealing common pathways and therapeutic targets.