DNAJC7 Antibody

What is DNAJC7 and why is it significant in neurodegenerative disease research?

DNAJC7 is a member of the DNAJ/HSP40 family of molecular chaperones that regulates protein folding and quality control. Its significance in neurodegenerative disease research stems from several critical findings:

• DNAJC7 specifically binds tau protein with nanomolar affinity and regulates tau aggregation in cooperation with Hsp70

• DNAJC7 gene mutations have been genetically linked to amyotrophic lateral sclerosis (ALS), a late-onset neurodegenerative disease characterized by motor neuron degeneration

• Knockout of DNAJC7 in cellular models has been shown to significantly increase tau seeding and aggregation, suggesting a protective role against pathological protein accumulation

The protein functions within the cellular quality control system where DNAJ dysfunction may result in aberrant folding of newly synthesized polypeptides or altered clearance of degraded proteins, contributing to disease pathology .

What are the typical experimental applications for DNAJC7 antibodies?

DNAJC7 antibodies can be utilized in multiple experimental contexts:

The antibody is particularly valuable for studying endogenous DNAJC7 in cellular models of protein aggregation disorders, examining chaperone-substrate interactions, and investigating the role of DNAJC7 in protein quality control pathways .

How can I confirm the specificity of a DNAJC7 antibody in my experimental system?

Confirming antibody specificity is crucial for obtaining reliable results. A methodological approach includes:

• Positive control validation: Use cell lines known to express DNAJC7 (most mammalian cells express this protein endogenously)

• Knockout validation: Compare antibody reactivity in wild-type cells versus DNAJC7 knockout cells (as described in the studies where CRISPR/Cas9 was used to generate DNAJC7 knockout lines)

• Recombinant protein detection: Test antibody against purified recombinant DNAJC7 protein

• Band size verification: Confirm detection at the expected molecular weight (approximately 62 kDa)

• Cross-reactivity assessment: If working across species, verify that the antibody recognizes the target in your specific experimental system, noting that the antibody described shows reactivity with human, mouse, and rat DNAJC7

Antibody validation is particularly important when studying protein interactions or when using the antibody for quantitative applications in disease models.

How can DNAJC7 antibodies be used to investigate tau aggregation mechanisms in neurodegenerative disease models?

DNAJC7 antibodies can be strategically employed to investigate tau aggregation through several methodological approaches:

• Co-immunoprecipitation studies: DNAJC7 antibodies can pull down protein complexes to analyze DNAJC7 interactions with tau and other chaperones like Hsp70. Research has demonstrated that DNAJC7 binds tau with nanomolar affinity and reduces tau aggregation in vitro .

• Clearance mechanism analysis: In cellular models, DNAJC7 knockout significantly impaired tau aggregate clearance, with ~40% of cells still containing aggregates after 5 days of tau repression compared to ~0% in control cells . DNAJC7 antibodies can help track this protein during clearance processes.

• Chaperone-mutant interaction studies: Compare wild-type versus mutant DNAJC7 binding to tau using antibody-based detection methods. For example, research has shown that disease-associated mutations in the J domain (JD) and substrate binding site of DNAJC7 abolished its protective activity against tau aggregation .

• Proximity ligation assays: Combine DNAJC7 antibodies with tau antibodies to visualize and quantify direct interactions in situ and how these may be altered in disease states.

• Immunofluorescence co-localization: Track the co-localization of DNAJC7 with tau aggregates in cellular models using confocal microscopy, as studies have shown DnaJC7 knockout increased inclusion density and decreased the quantity of diffuse tau .

When designing such experiments, it's critical to consider the specificity of the DNAJC7 antibody for detecting different conformational states of the protein and its potential epitope accessibility in protein complexes.

What methodological approaches can address the apparent discrepancy between DNAJC7 mRNA and protein levels in disease states?

Research has revealed an interesting discrepancy between DNAJC7 mRNA and protein levels, particularly regarding post-translational modifications. In renal cell carcinoma studies, while no significant difference was found in DNAJC7 mRNA levels between cancerous and pericancerous tissues, polyglutamylated-DNAJC7 protein was significantly elevated in cancer samples . This suggests post-translational regulation is critical for DNAJC7 function.

To investigate this phenomenon:

• Parallel RNA and protein quantification: Simultaneously measure DNAJC7 mRNA using qRT-PCR and protein using Western blotting with DNAJC7 antibodies in the same samples to establish correlation patterns.

• Post-translational modification detection: Employ a two-step approach:

  • Immunoprecipitate with DNAJC7 antibody

  • Probe with modification-specific antibodies (e.g., GT335 for polyglutamylation)

• Protein stability assessment: Use cycloheximide chase experiments with DNAJC7 antibody detection to determine if disease states alter protein half-life without affecting transcription.

• Translational efficiency analysis: Combine polysome profiling with DNAJC7 mRNA quantification to assess translational control.

• Proteasomal degradation studies: Use proteasome inhibitors and DNAJC7 antibodies to determine if differential degradation explains the mRNA-protein discrepancy.

This methodological framework can help researchers understand the complex regulation of DNAJC7 in disease contexts and potentially explain why transcriptional measurements alone may not reflect functional protein levels.

How can immunoassay-based detection of polyglutamylated DNAJC7 be optimized for biomarker applications?

Polyglutamylated DNAJC7 has shown promise as a biomarker for renal cell carcinoma (RCC) with remarkable sensitivity and specificity. Research has demonstrated that electrochemiluminescence immunoassay (ECLIA) using GT335 antibody can detect polyglutamylated DNAJC7 in serum with 94.3% sensitivity and 99.1% specificity for early-stage RCC detection . To optimize such assays:

• Antibody selection strategy:

  • Primary capture antibody: Use anti-DNAJC7 antibody with high specificity and affinity

  • Detection antibody: Employ modification-specific antibody (e.g., GT335 for polyglutamylation)

• Sample preparation optimization:

  • Standardize collection protocols to minimize pre-analytical variables

  • Develop consistent serum processing methods to preserve post-translational modifications

  • Consider enrichment steps if detection sensitivity is limited

• Assay validation parameters:

  • Establish calibration curves using recombinant polyglutamylated DNAJC7

  • Determine lower limit of detection (LLOD) and quantification (LLOQ)

  • Assess precision through intra- and inter-assay coefficient of variation (%CV)

  • Evaluate recovery and linearity across physiologically relevant concentrations

• Clinical cutoff determination:

  • Use ROC analysis to establish optimal cutoff values (research has identified RLU values between 3642-4062 as effective cutoff criteria)

  • Calculate sensitivity and specificity at various cutoff points for different disease stages

• Cross-validation with multiple cohorts:

  • Test cohort validation followed by larger validation cohort analysis

  • Account for disease heterogeneity by staging (e.g., TNM staging for RCC)

This methodological approach supports reliable detection of polyglutamylated DNAJC7 as a biomarker, with research showing potential for distinguishing not only cancer from non-cancer but also different stages of disease progression .

What controls should be included when using DNAJC7 antibodies in studies of neurodegenerative disease mechanisms?

Robust experimental design requires appropriate controls to ensure reliable interpretations of DNAJC7's role in neurodegenerative diseases:

• Genetic controls:

  • DNAJC7 knockout cells: Essential for antibody validation and functional studies. Research has shown DNAJC7 KO significantly affects tau aggregate clearance

  • Rescue experiments: Expression of gRNA-resistant DNAJC7 constructs in knockout cells should restore normal phenotype, confirming specificity of observed effects

  • Mutant controls: Include disease-associated DNAJC7 mutants (e.g., HPD motif mutations that inhibit Hsp70 binding, as well as ALS-associated mutations like R412W, R425K)

• Technical controls:

  • Antibody concentration gradients to determine optimal working dilutions

  • Secondary antibody-only controls to assess non-specific binding

  • Loading controls appropriate for subcellular fractions being analyzed

  • Pre-adsorption controls using recombinant DNAJC7 to confirm antibody specificity

• Experimental paradigm controls:

  • Temporal controls: Include multiple timepoints for aggregation/clearance experiments (research shows differential effects at 24 vs. 48 hours for seeding experiments)

  • Concentration gradients: Use dose-response curves for seed concentration when studying aggregation (as performed in the tau seeding studies)

  • Alternative JDP family members: Include other J-domain proteins as specificity controls (studies showed that among 50 JDP knockout lines, only DNAJC7 and DnaJB6 significantly affected tau seeding)

These controls are critical for distinguishing between specific DNAJC7-mediated effects and potential artifacts, particularly when investigating complex processes like protein aggregation and clearance in neurodegenerative disease models.

How can I optimize immunoprecipitation protocols using DNAJC7 antibodies to study chaperone-substrate interactions?

Optimizing immunoprecipitation (IP) protocols for DNAJC7 requires careful consideration of multiple factors to preserve physiologically relevant interactions:

• Lysis buffer optimization:

  • For studying weak or transient interactions: Use crosslinking agents (e.g., DSP or formaldehyde) prior to lysis

  • For studying stable interactions: Use mild non-ionic detergents (0.5-1% NP-40 or Triton X-100)

  • Include protease inhibitors, phosphatase inhibitors, and deubiquitinase inhibitors

  • For post-translational modification studies: Include specific inhibitors (e.g., deglutamylase inhibitors when studying polyglutamylated DNAJC7)

• Antibody selection and coupling:

  • Test multiple DNAJC7 antibody clones for IP efficiency

  • Pre-clear lysates to reduce non-specific binding

  • Consider using antibody-conjugated magnetic beads for cleaner IP results

  • For sequential IP (e.g., first DNAJC7 then tau), optimize elution conditions that preserve the integrity of the first immunoprecipitated complex

• Co-factor considerations:

  • Include ATP (1-5 mM) to stabilize certain chaperone-substrate interactions

  • Consider adding magnesium (required for ATPase activity)

  • Test different salt concentrations to optimize stringency

• Specialized applications:

  • For studying DNAJC7-Hsp70 interactions: IP in the presence and absence of client proteins

  • For tau interaction studies: Compare binding efficiency with different tau species (e.g., monomeric vs. pre-aggregated)

  • For capturing transient interactions: Consider proximity-dependent biotinylation approaches (BioID or TurboID) as complementary methods

• Validation approaches:

  • Perform reciprocal IPs (e.g., IP with DNAJC7 antibody followed by tau detection, and vice versa)

  • Include wild-type and mutant DNAJC7 controls (e.g., HPD motif mutants) to confirm specificity

  • Use mass spectrometry to identify additional interacting partners in an unbiased manner

This methodological framework will help capture physiologically relevant interactions while minimizing artifacts commonly encountered in chaperone interaction studies.

What are the critical variables to control when using DNAJC7 antibodies in tau seeding experiments?

Tau seeding experiments with DNAJC7 antibodies require careful control of multiple variables to ensure reproducibility and meaningful results:

• Seed preparation standardization:

  • Source consistency: Use seeds from the same brain region or cell type

  • Sonication protocol: Standardize duration, amplitude, and pulse parameters

  • Size fractionation: Consider ultracentrifugation to isolate specific aggregate species

  • Quantification method: Use consistent protein concentration measurement techniques

• Cellular model considerations:

  • DNAJC7 expression levels: Quantify baseline expression in your model system

  • Tau expression: Control for tau isoform and expression level

  • Seeding efficiency baseline: Establish dose-response curves for your specific model

  • Passage number: Use cells within a defined passage range to minimize phenotypic drift

• Experimental timing factors:

  • Seed exposure duration: Research shows differential seeding at 24 vs. 48 hours

  • Analysis timepoints: No significant seeding observed at 6 and 12 hours in studies, with effects becoming apparent at 24 and 48 hours

  • Expression kinetics: For inducible systems, standardize induction and repression protocols

• Quantification parameters:

  • Flow cytometry gating strategy: Define consistent gates for aggregate-positive cells

  • Microscopy settings: Standardize exposure times, gain settings, and threshold values

  • Image analysis: Use automated, unbiased quantification algorithms

• Perturbation controls:

  • DNAJC7 knockdown/knockout validation: Confirm protein reduction via Western blot

  • Rescue experiments: Use expression constructs resistant to knockdown/knockout

  • Mutant variants: Include DNAJC7 mutants that affect Hsp70 binding (HPQ) or substrate recognition as functional controls

Research has shown that even partial knockout of DNAJC7 significantly increases tau seeding , emphasizing the importance of quantitative validation of DNAJC7 levels in experimental systems.

How can DNAJC7 antibodies be utilized to investigate the link between polyglutamylation and disease biomarkers?

Research has identified polyglutamylated DNAJC7 as a promising biomarker for renal cell carcinoma . This finding opens avenues for investigating similar modifications in other diseases using antibody-based approaches:

• Modification-specific detection strategies:

  • Two-step immunodetection: First capture with DNAJC7 antibody, then probe with polyglutamylation-specific antibody (GT335)

  • Development of modification-site-specific antibodies recognizing both DNAJC7 and its polyglutamylation simultaneously

  • Quantitative comparison between total DNAJC7 (detected by standard antibodies) and modified forms

• Cross-disease investigation methodology:

  • Apply similar immunoassay approaches to analyze polyglutamylated DNAJC7 in neurodegenerative disease samples (especially given DNAJC7's role in tau pathology)

  • Compare polyglutamylation patterns across cancer types beyond renal cell carcinoma

  • Correlate polyglutamylation levels with disease progression markers in longitudinal studies

• Mechanistic study approaches:

  • Use DNAJC7 antibodies to immunoprecipitate the protein from disease vs. normal tissues for:

    • Mass spectrometry analysis of all post-translational modifications

    • Enzymatic activity assays to determine functional consequences of polyglutamylation

    • Binding partner analysis to identify differential interactions with modified DNAJC7

• Technical optimization for detection:

  • Based on previous studies, electrochemiluminescence immunoassay (ECLIA) using GT335 antibody showed excellent sensitivity and specificity for polyglutamylated DNAJC7

  • Standardize cutoff values: Research identified RLU values between 3642-4062 as effective thresholds

  • Consider multivariate models combining polyglutamylated DNAJC7 with other biomarkers

This methodological framework leverages DNAJC7 antibodies to explore a promising intersection between protein quality control, post-translational modifications, and disease biomarkers.

What approaches can be used to study the mechanistic relationship between DNAJC7 mutations and ALS pathogenesis?

DNAJC7 has been genetically linked to amyotrophic lateral sclerosis (ALS) , but the mechanistic details require further investigation. DNAJC7 antibodies can facilitate this research through several approaches:

• Mutation-specific functional analysis:

  • Compare wild-type and mutant DNAJC7 localization using immunocytochemistry

  • Analyze co-immunoprecipitation efficiency of disease-associated mutants with Hsp70 and client proteins

  • Study the impact of mutations on DNAJC7's ability to prevent protein aggregation in cellular models

• ALS-relevant substrate identification:

  • Use DNAJC7 antibodies for immunoprecipitation followed by mass spectrometry to identify ALS-relevant client proteins

  • Compare client binding profiles between wild-type and ALS-associated DNAJC7 mutants

  • Validate identified interactions using reciprocal co-immunoprecipitation

• Motor neuron-specific investigation:

  • Apply DNAJC7 antibodies to study protein expression and localization in motor neuron cultures or tissue

  • Analyze changes in DNAJC7 levels or localization during ALS progression in model systems

  • Examine post-translational modifications of DNAJC7 in ALS contexts

• Structure-function relationship studies:

  • Use antibodies recognizing different domains to assess structural changes in mutant DNAJC7

  • Combine with biochemical assays to correlate structural alterations with functional deficits

  • Research has shown ALS-associated mutations may inhibit DNAJC7 interaction with other chaperones (Hsp70 and Hsp90), impairing substrate handoff

• Therapeutic targeting assessment:

  • Use DNAJC7 antibodies to monitor the effects of small molecules designed to modulate chaperone function

  • Evaluate the impact of therapies on DNAJC7-client interactions in disease models

  • Track changes in DNAJC7 levels or activity in response to experimental treatments

This approach leverages both the diagnostic potential of DNAJC7 antibodies and their utility in mechanistic studies relevant to ALS pathogenesis.

How can researchers apply DNAJC7 antibodies to investigate the specificity of chaperone-mediated protein quality control?

Research has demonstrated that DNAJC7 plays a specific role in tau protein quality control that is not shared by most other J-domain proteins (JDPs) . Investigating this specificity can provide insights into targeted approaches for neurodegenerative diseases:

• Comparative chaperone interaction mapping:

  • Use DNAJC7 antibodies alongside antibodies against other JDPs in co-immunoprecipitation experiments

  • Analyze differential binding partners by mass spectrometry

  • Research has shown that among 50 JDP knockout lines, only DNAJC7 and DnaJB6 significantly affected tau seeding

• Domain-specific function analysis:

  • Generate domain-selective DNAJC7 antibodies targeting:

    • J-domain (implicated in Hsp70 interaction)

    • TPR domains (involved in substrate recognition)

    • C-terminal regions (potential regulatory functions)

  • Combine with domain deletion/mutation constructs to correlate structure with function

  • Research has shown that DNAJC7 binds tau via its TPR2B domain and engages Hsp70 to stabilize inert tau conformations

• Client protein selectivity assessment:

  • Use DNAJC7 antibodies to isolate chaperone-client complexes

  • Compare binding affinities across different disease-associated proteins (tau, α-synuclein, TDP-43, etc.)

  • Correlate binding preferences with structural features of client proteins

• Functional redundancy investigation:

  • Apply DNAJC7 antibodies in models with manipulated levels of multiple chaperones

  • Assess compensatory responses following DNAJC7 knockout or overexpression

  • Study competitive vs. cooperative interactions between different chaperone systems

• Quantitative proteomics approach:

  • Use DNAJC7 antibodies for immunoprecipitation followed by quantitative proteomics

  • Apply SILAC or TMT labeling to compare client specificity under different cellular stresses

  • Identify condition-specific changes in DNAJC7-client interactions

This methodological framework leverages antibodies to investigate both the specificity and redundancy within chaperone networks, potentially identifying therapeutic windows where modulating DNAJC7 could selectively affect disease-relevant substrates without disrupting essential protein quality control functions.

What are the future directions for DNAJC7 antibody applications in disease research?

Research utilizing DNAJC7 antibodies has already revealed significant insights into protein quality control mechanisms and disease biomarkers. Future directions will likely expand these applications in several key areas:

• Biomarker development beyond RCC: Given the success of polyglutamylated DNAJC7 as a renal cell carcinoma biomarker , expansion to other diseases including neurodegenerative conditions where DNAJC7 plays a role in protein aggregation represents a promising direction.

• Therapeutic target validation: DNAJC7 antibodies will be invaluable for validating therapeutic approaches aimed at enhancing chaperone function or preventing pathological protein aggregation.

• Post-translational modification mapping: Comprehensive characterization of disease-specific modifications of DNAJC7 could reveal regulatory mechanisms and new biomarker opportunities.

• Patient stratification tools: Development of antibody-based assays to identify patient subgroups based on DNAJC7 function or modification status could enable personalized medicine approaches.

• Integration with emerging technologies: Combining DNAJC7 antibodies with single-cell analysis, spatial proteomics, and in vivo imaging could provide unprecedented insights into chaperone biology in complex tissues.

These future applications will require continued refinement of antibody specificity, sensitivity, and application protocols, but promise to expand our understanding of protein quality control in health and disease while potentially yielding new diagnostic and therapeutic approaches.

What considerations should researchers keep in mind when selecting DNAJC7 antibodies for specific applications?

Selecting the appropriate DNAJC7 antibody requires careful consideration of several factors to ensure experimental success:

• Epitope location and accessibility:

  • For studying protein interactions: Choose antibodies targeting non-interface regions

  • For detecting post-translational modifications: Select antibodies whose epitopes don't overlap with modification sites

  • For immunoprecipitation: Consider antibodies recognizing native epitopes

• Validation status for specific applications:

  • Confirm antibody has been validated for your application (WB, IP, ICC, etc.)

  • Check species reactivity matches your experimental system (human, mouse, rat)

  • Review validation data including knockout controls when available

• Clone characteristics:

  • Monoclonal antibodies offer consistency across experiments

  • Polyclonal antibodies may provide better detection through recognition of multiple epitopes

  • Consider using antibody combinations for comprehensive analysis

• Application-specific optimizations:

  • Western blotting: Typical dilution reported is 1:1000

  • Immunoprecipitation: May require higher concentrations

  • Immunohistochemistry: Fixation method may affect epitope accessibility

• Experimental design alignment:

  • For functional studies: Select antibodies that don't interfere with critical protein interactions

  • For quantitative applications: Choose antibodies with demonstrated linear response range

  • For multiplexing: Consider host species compatibility with other antibodies in your panel