park7 Antibody

What is PARK7/DJ-1 and why is it significant in neurodegenerative research?

PARK7/DJ-1 is a multifunctional protein belonging to the ThiJ/Pfp1/DJ-1 superfamily that functions as a molecular chaperone, redox sensor, and antioxidant protein. It plays a critical protective role against oxidative stress and cell death. PARK7's significance stems from its association with autosomal recessive, early-onset Parkinson's disease, where mutations impair transcriptional coactivator function, rendering dopaminergic neurons vulnerable to apoptosis . Recent research has revealed that PARK7 prevents damage to proteins and metabolites caused by the glycolytic intermediate 1,3-bisphosphoglycerate, suggesting a novel mechanism potentially contributing to Parkinson's disease pathogenesis .

What cellular localization patterns does PARK7/DJ-1 exhibit?

PARK7/DJ-1 has a complex localization pattern that depends on cellular conditions. Under normal conditions, it is located predominantly in the cytoplasm and, to a lesser extent, in the nucleus and mitochondria . During oxidative stress, it translocates first to the mitochondria and subsequently to the nucleus, where it exerts an increased cytoprotective effect . Importantly, PARK7 has been detected in tau inclusions in brains from neurodegenerative disease patients. When conducting immunocytochemistry experiments, researchers should observe this differential localization, which can serve as an internal validation of antibody performance and cellular stress conditions .

What applications are PARK7/DJ-1 antibodies validated for?

PARK7/DJ-1 antibodies have been validated for multiple applications including:

Most antibodies require optimization for specific experimental conditions .

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

Validation of PARK7/DJ-1 antibodies should follow a multi-step approach:

• Knockout validation: Use PARK7 knockout cell lines (such as PARK7-KO HEK293T cells) to confirm absence of signal in Western blots. Multiple sources show specific bands at approximately 23 kDa in parental cell lines that are absent in knockout lines .

• Recombinant protein controls: Include purified recombinant PARK7 as a positive control in Western blots to confirm correct molecular weight detection.

• Cross-species reactivity testing: Many PARK7 antibodies show differential reactivity across species. For example, the 4H4 clone detects human PARK7 but not rodent forms, while other antibodies detect human, mouse, and rat PARK7 .

• Multiple application validation: Confirm antibody performance across different applications (WB, ICC, IHC) to ensure consistent detection.

• Epitope mapping: Consider the epitope location when interpreting results, as some antibodies target regions that may be masked during protein interactions or post-translational modifications .

What are the critical differences in PARK7/DJ-1 detection between human and rodent samples?

Significant species-specific differences exist in PARK7/DJ-1 detection:

• Expression patterns: In human brain tissue, PARK7 shows marked astrocytic expression with weak or absent neuronal labeling. In contrast, rat brain exhibits ubiquitous neuronal expression with low astrocytic expression .

• Antibody cross-reactivity: Some antibodies like the 4H4 clone (NBP1-92715) specifically detect human PARK7 but not mouse or rat PARK7, showing a 21 kDa band only in human cell lines . Other antibodies (such as MAB39951) detect PARK7 across human, mouse, and rat samples .

• Molecular weight variations: Human PARK7 typically appears at 21-23 kDa, while observed molecular weights may vary slightly between species or in different gel systems (Simple Western may show bands at ~26-28 kDa) .

• Knock-out controls: When working with rodent models, researchers should carefully select antibodies validated for the specific species and include appropriate controls .

When designing cross-species experiments, researchers should verify antibody reactivity with each species and be cautious about interpreting expression differences .

How does oxidative stress affect PARK7/DJ-1 detection with antibodies?

Oxidative stress significantly impacts PARK7/DJ-1 detection due to its redox-sensitive nature:

• Oxidation of cysteine residues: PARK7 can be oxidized at Cys46, Cys53, and Cys106. While PARK7 can undergo oxidation-reduction cycling, oxidation of all three Cys residues is irreversible . This oxidation may affect epitope accessibility for certain antibodies.

• Subcellular translocation: Under oxidative stress, PARK7 translocates from the cytoplasm to mitochondria and subsequently to the nucleus, changing its detection pattern in immunocytochemistry/immunofluorescence experiments .

• Protein interactions: Oxidative stress enhances PARK7's interaction with other proteins like HSPA5 (an ER-resident chaperone), which may mask certain epitopes .

• Experimental considerations:

  • Include both reducing and non-reducing conditions in Western blots to capture different oxidation states

  • Use subcellular fractionation to track PARK7 movement during oxidative stress

  • Consider using antibodies that target different epitopes to ensure detection of all PARK7 forms

  • Include positive controls using cells treated with oxidative stress inducers (e.g., TNFSF10 has been shown to enhance R-HSPA5 formation and interaction with PARK7)

What methodological approaches can detect PARK7/DJ-1 interactions with other proteins?

Several approaches can effectively detect PARK7/DJ-1 protein interactions:

• Co-immunoprecipitation (co-IP):

  • Use 0.5-4.0 μg of PARK7 antibody for 1.0-3.0 mg of total protein lysate

  • Both endogenous and overexpressed systems have successfully shown PARK7 interactions

  • FLAG-tagged PARK7 can be used with anti-FLAG antibody resin for cleaner pull-downs

  • Detection of interacting partners like HSPA5 requires specific antibodies against those proteins

• Proximity ligation assays: For detecting in situ protein interactions in fixed cells or tissues

• GST affinity-isolation assays:

  • Using bacterially purified GST-PARK7 has successfully demonstrated interactions with proteins like R-HSPA5

  • This approach allows for controlled in vitro conditions

• Mass spectrometry following IP:

  • LC-MS/MS analysis of co-purified proteins has identified PARK7 interaction partners

  • Treatment with stress inducers like TNFSF10 can reveal condition-specific interactions

• Cellular fractionation:

  • Differential centrifugation to separate cytosol, mitochondria, and ER fractions

  • Western blot analysis of fractions can reveal compartment-specific interactions

Different stress conditions significantly affect PARK7's interactome, so experimental design should account for cellular state .

How can researchers study PARK7/DJ-1 modifications using specific antibodies?

PARK7/DJ-1 undergoes various modifications that can be studied using specific methodological approaches:

• Phosphoglycerate and glycerate modifications:

  • Recent research has shown PARK7 prevents damage caused by phosphoglycerate and glycerate modifications

  • Proteolytic digests from tissues analyzed by nano-LC-MS with phosphopeptide-enrichment protocols can identify modified peptides

  • PARK7 knockout samples show significantly higher levels of these modifications

  • Expression of wild-type PARK7 but not mutant forms in knockout cells reduces these modifications

• Oxidative modifications:

  • Cysteine oxidation (particularly at Cys106) is functionally significant

  • Reducing vs. non-reducing conditions in Western blots can help distinguish oxidized forms

  • Subcellular fractionation combined with Western blotting can track oxidation-induced translocation

• N-terminal modifications:

  • PARK7 interactions can be affected by N-terminal arginylation of partner proteins

  • Antibodies specific to modified forms (e.g., R-HSPA5) are necessary to detect these interactions

• Methodological approaches:

  • Pulse-chase experiments with isotope-labeled precursors can track modification dynamics

  • In vitro reconstitution systems with recombinant PARK7 and modification sources allow controlled studies

  • Mutational analysis of key residues can confirm modification sites and functional significance

What are common issues with PARK7/DJ-1 antibodies and how can they be addressed?

Common issues with PARK7/DJ-1 antibodies include:

• Multiple bands in Western blots:

  • Verify antibody specificity using knockout controls

  • Check for degradation products by using fresh samples and protease inhibitors

  • Test different reducing conditions as PARK7 is redox-sensitive

  • Different molecular weights may be observed (21-28 kDa) depending on gel systems and sample preparation

• Weak or absent signal:

  • Optimize antibody concentration (typical ranges: 0.2-1.0 μg/mL for Western blot)

  • Ensure appropriate blocking (some antibodies work best with specific blocking buffers)

  • For IHC/ICC, test different antigen retrieval methods (heat-induced epitope retrieval has been successful)

  • Consider species specificity issues, as some antibodies don't cross-react with all species

• High background:

  • Increase washing steps (duration and number)

  • Titrate primary antibody to determine optimal concentration

  • Test different blocking agents (BSA vs. milk)

  • For immunofluorescence, include an autofluorescence quenching step

• Inconsistent results between experiments:

  • Use standardized lysate preparation methods

  • Aliquot antibodies to avoid freeze-thaw cycles

  • Include positive controls in each experiment

  • Maintain consistent exposure times for imaging or development

How should PARK7/DJ-1 antibodies be stored and handled for optimal performance?

Proper storage and handling of PARK7/DJ-1 antibodies is critical for maintaining performance:

• Storage conditions:

  • Store at -20°C to -70°C for long-term storage (most antibodies are stable for at least 12 months)

  • For reconstituted lyophilized antibodies, store at 2-8°C for up to 1 month or aliquot and store at -20°C for up to 6 months

  • Avoid repeated freeze-thaw cycles by preparing small working aliquots

• Reconstitution:

  • For lyophilized antibodies, reconstitute in sterile water or buffer as recommended by manufacturer

  • Some antibodies are supplied in PBS with preservatives like sodium azide and/or glycerol

• Working dilutions:

  • Prepare fresh working dilutions on the day of experiment

  • For Western blot applications, dilution ranges from 1:1000-1:10000 are typical

  • For immunocytochemistry, dilutions of 1:125-1:500 are common

• Shipping and handling:

  • Most antibodies are shipped on blue ice or at 4°C

  • Upon receipt, immediately transfer to recommended storage conditions

  • Check for any precipitates before use; if present, gently mix or centrifuge

• Stability considerations:

  • Most antibodies remain stable for 1 year from date of receipt when stored properly

  • Some formulations contain stabilizing proteins (like BSA) or preservatives that extend shelf life

What factors should be considered when selecting between monoclonal and polyclonal PARK7/DJ-1 antibodies?

The choice between monoclonal and polyclonal PARK7/DJ-1 antibodies depends on several research considerations:

For validation studies, using both monoclonal and polyclonal antibodies targeting different epitopes can provide complementary data and stronger confirmation of results .

What controls are essential when studying PARK7/DJ-1 in neurodegenerative disease models?

When studying PARK7/DJ-1 in neurodegenerative disease models, several essential controls should be included:

• Knockout/knockdown validation:

  • Use PARK7 knockout cell lines to confirm antibody specificity

  • PARK7-KO HEK293T cells show complete absence of the 23 kDa band present in parental lines

  • For in vivo models, include samples from PARK7 knockout animals when available

• Loading controls:

  • Include housekeeping proteins like GAPDH to normalize expression levels

  • When studying subcellular localization, include markers for each compartment (cytosolic, nuclear, mitochondrial)

• Recombinant protein standards:

  • Include purified recombinant PARK7 as positive control for Western blots

  • Use for standard curves in quantitative applications like ELISA

• Disease-relevant controls:

  • Compare samples from sporadic and familial Parkinson's disease

  • Include age-matched controls when studying neurodegenerative conditions

  • Consider co-staining for pathological markers (Lewy bodies, tau aggregates)

• Oxidative stress controls:

  • Include samples treated with oxidative stress inducers as positive controls

  • Compare reducing and non-reducing conditions to detect redox-sensitive changes

• Species-specific considerations:

  • Be aware that human PARK7 shows stronger astrocytic than neuronal expression, while rat PARK7 shows the opposite pattern

  • Some antibodies do not cross-react between human and rodent samples

How can PARK7/DJ-1 antibodies be used to study its role in preventing metabolite and protein damage?

Recent research has revealed PARK7/DJ-1's role in preventing damage caused by reactive glycolytic intermediates, which can be studied using these methodological approaches:

• Detection of phosphoglycerate and glycerate modifications:

  • Phosphopeptide-enrichment protocols significantly improve identification of phosphoglycerate-modified peptides

  • Nano-LC-MS analysis of proteolytic digests can identify modified peptides (62 phosphoglycerate-modified peptides from 51 proteins were identified in PARK7 knockout mouse brain)

  • Compare PARK7 knockout samples with wild-type and rescue experiments to confirm PARK7's protective role

• In vitro reconstitution systems:

  • Reactions producing 1,3-BPG via PGK with GAPDH present in reaction mixtures

  • Analysis of protein modifications reveals phosphoglycerate-modifications that are reduced in the presence of PARK7

  • Include recombinant PARK7 or mutant variants to test structure-function relationships

• Pulse-chase experiments:

  • Use isotope-labeled (13C) glycine to track the formation of newly synthesized glycerate-adducts

  • Induction of PARK7 expression reduced the m+3 fraction by more than twofold, whereas mutant PARK7 showed no effect

  • This approach demonstrates PARK7 prevents formation rather than removes existing modifications

• Methodological controls:

  • Verify that PARK7 doesn't directly affect 1,3-BPG levels

  • Include reactions with and without PARK7 to quantify its protective effect

  • Test PARK7 mutants to identify critical residues for this function

What new methodological approaches are emerging for studying PARK7/DJ-1 in cellular models?

Emerging methodological approaches for studying PARK7/DJ-1 include:

• CRISPR-Cas9 engineered cellular models:

  • PARK7 knockout cell lines provide cleaner backgrounds for reconstitution experiments

  • Precise editing allows introduction of disease-associated mutations

  • Knockout validation demonstrated with multiple antibodies shows complete absence of signal in Western blots

• Live-cell imaging techniques:

  • Fluorescently tagged PARK7 allows real-time monitoring of subcellular translocation during stress

  • FRET-based sensors can detect PARK7 interactions with partner proteins

  • Photoactivatable constructs enable tracking of specific protein populations

• Proximity-dependent labeling:

  • BioID or APEX2 fusions to PARK7 allow identification of proximal proteins in living cells

  • Particularly useful for capturing transient or weak interactions in specific cellular compartments

• Single-cell analysis:

  • Combining immunofluorescence with single-cell RNA-seq provides correlations between PARK7 protein levels and transcriptional responses

  • Helps understand cell-to-cell variability in PARK7 function

• 3D cellular models:

  • Organoids derived from patient iPSCs with PARK7 mutations

  • More physiologically relevant than traditional 2D cultures

  • Can be analyzed with immunohistochemistry using validated PARK7 antibodies

How do PARK7/DJ-1 antibodies contribute to understanding its role in neurodegeneration?

PARK7/DJ-1 antibodies have revealed several aspects of its role in neurodegeneration:

• Cell-type specific expression patterns:

  • Immunohistochemistry studies show PARK7 is predominantly expressed in astrocytes rather than neurons in human brain tissue, whereas in rat brain it shows ubiquitous neuronal expression with low astrocytic presence

  • This suggests species-specific differences in PARK7 function that may impact translational research

• Lack of Lewy body association:

  • Antibody studies revealed that PARK7 does not label Lewy bodies or Lewy neurites

  • This distinguishes PARK7-linked Parkinson's disease from other forms

• Subcellular localization changes:

  • Under oxidative stress, PARK7 translocates from cytoplasm to mitochondria and nucleus

  • This translocation can be tracked using immunofluorescence with PARK7 antibodies

• Interaction with stress response systems:

  • Immunoprecipitation studies revealed PARK7 interacts with chaperones including HSPA5, HSPA8/HSC70, HSPA1A, and HSP90AA1

  • These interactions are enhanced under oxidative stress conditions

• Protection from glycolytic intermediate damage:

  • Comparative studies between wild-type and PARK7 knockout samples revealed PARK7 prevents damage caused by 1,3-BPG

  • This suggests a novel mechanism potentially contributing to neurodegeneration when PARK7 is mutated or absent