DNAJC15 Antibody, HRP conjugated

What is DNAJC15 and what are its primary cellular functions?

DNAJC15, also known as methylation-controlled J protein (MCJ) or heat shock protein 40 (HSP40), is a mitochondrial cochaperone that localizes to the inner mitochondrial membrane. This protein plays crucial roles in cellular physiology through several mechanisms:

• Regulation of mitochondrial respiration: DNAJC15 functions as a negative regulator of the respiratory chain by interacting preferentially with complex I of the electron transfer chain .

• Protein folding assistance: As part of the HSP40 family, it assists in proper protein folding and prevents aggregation under stressful conditions .

• Suppression of supercomplex formation: DNAJC15 impairs the formation of respiratory chain supercomplexes, affecting oxidative phosphorylation efficiency .

• Metabolic regulation: Loss of DNAJC15 leads to increased complex I activity, enhanced mitochondrial membrane potential, and elevated ATP production .

• Stress response mediation: It plays a role in cellular stress response mechanisms, particularly in contexts involving oxidative stress .

Recent research has also revealed its involvement in lipid metabolism and ferroptosis susceptibility, with implications for cancer therapy response, particularly in ovarian cancer .

What is the expected molecular weight of DNAJC15 in Western blot applications?

When using HRP-conjugated DNAJC15 antibodies in Western blot applications, researchers should expect to observe bands at approximately 18-20 kDa, which is slightly higher than the calculated molecular weight of 16 kDa based on the 150 amino acid sequence . This discrepancy between calculated and observed molecular weights is common for many proteins due to post-translational modifications or structural characteristics affecting migration patterns during electrophoresis.

For experimental planning purposes:

• Primary band: 18-20 kDa (native DNAJC15)

• Calculated molecular weight: 16 kDa (based on amino acid sequence)

• Sample preparation: Careful extraction of mitochondrial fractions may be necessary for optimal detection

Researchers should validate their specific antibody with positive controls such as mouse liver tissue or human brain tissue, which have been documented to show reliable DNAJC15 expression .

What tissue and cell types are recommended as positive controls for DNAJC15 antibody validation?

Based on validated experimental data, the following samples are recommended as positive controls when working with DNAJC15 antibodies:

When establishing a new experimental system, it is advisable to include at least one of these validated positive controls alongside your samples of interest to confirm proper antibody functionality .

What applications are HRP-conjugated DNAJC15 antibodies suitable for?

HRP-conjugated DNAJC15 antibodies are particularly well-suited for enzyme-linked detection methods where the horseradish peroxidase conjugation provides a direct detection mechanism. The most appropriate applications include:

• Western blotting: Optimal dilution ranges typically fall between 1:1000-1:4000 for standard DNAJC15 antibodies, but may need optimization for HRP-conjugated versions .

• Enzyme-linked immunosorbent assay (ELISA): Direct detection without secondary antibodies.

• Immunohistochemistry: Particularly useful for formalin-fixed, paraffin-embedded tissues with dilutions of 1:50-1:500 being typical starting points .

• In-cell Western assays: For quantitative analysis of expression in cultured cells.

How should samples be prepared for optimal DNAJC15 detection?

Optimal sample preparation for DNAJC15 detection requires careful consideration of its mitochondrial localization. The following protocol recommendations can maximize detection sensitivity:

For Western blot applications:

• Extract cell or tissue lysates using buffers containing mild detergents (0.5-1% Triton X-100 or NP-40).

• Include protease inhibitors to prevent degradation.

• Consider mitochondrial enrichment protocols for low-abundance samples.

• Denature samples at 95°C for 5 minutes in Laemmli buffer containing DTT or β-mercaptoethanol.

• Load 20-50 μg of total protein per lane for standard detection.

For immunohistochemistry:

• Fix tissues with 10% neutral buffered formalin.

• Perform antigen retrieval using TE buffer at pH 9.0 (preferred) or citrate buffer at pH 6.0 as an alternative .

• Block endogenous peroxidase activity if using HRP-conjugated antibodies.

• Include permeabilization steps to ensure antibody access to mitochondrial structures.

For maximum sensitivity, especially when investigating tissues with potentially low DNAJC15 expression, mitochondrial fraction isolation prior to analysis can significantly improve signal-to-noise ratios .

How can DNAJC15 antibodies be optimized for studying its role in mitochondrial respiratory complex interactions?

Studying DNAJC15's interactions with respiratory complexes, particularly complex I, requires specialized approaches:

• Blue Native PAGE Optimization:

  • Solubilize mitochondrial membranes with digitonin (1-2%) to preserve native protein complexes.

  • Perform Blue Native PAGE followed by Western blot with HRP-conjugated DNAJC15 antibodies.

  • Consider second-dimension SDS-PAGE to separate complex components.

  • Start with 1:500 antibody dilution and optimize based on signal intensity.

• Proximity Ligation Assay (PLA) Protocol:

  • Fix cells with 4% paraformaldehyde.

  • Permeabilize with 0.1% Triton X-100.

  • Use DNAJC15 antibody alongside antibodies against complex I subunits (such as NDUFS1).

  • Employ PLA probes and detection reagents according to manufacturer protocols.

  • This approach allows visualization of in situ interactions between DNAJC15 and complex I.

• Co-immunoprecipitation Strategy:

  • Solubilize mitochondrial membranes with mild detergents (0.5-1% digitonin).

  • Perform immunoprecipitation with unconjugated DNAJC15 antibodies.

  • Analyze precipitates for complex I components by Western blot.

  • For reverse co-IP, immunoprecipitate with complex I antibodies and probe for DNAJC15.

These approaches have been instrumental in establishing DNAJC15's preferential interaction with complex I of the electron transport chain and its role in regulating supercomplex formation . When analyzing results, remember that DNAJC15 functions as a negative regulator of respiratory chain activity, so interaction data should be interpreted in this context .

What methodological approaches can be used to investigate DNAJC15's role in ferroptosis using HRP-conjugated antibodies?

Recent research has revealed DNAJC15's involvement in promoting ferroptosis in cancer cells, particularly in the context of chemotherapy resistance . The following methodological approaches can help investigate this phenomenon:

• Inducible Expression System Analysis:

  • Establish cell lines with inducible DNAJC15 expression.

  • Monitor DNAJC15 expression levels via Western blot with HRP-conjugated antibodies.

  • Measure lipid peroxidation using C11-BODIPY or TBARS assays.

  • Quantify ferroptosis markers (GPX4 reduction, iron accumulation) following DNAJC15 induction.

  • Test ferroptosis inhibitors (Ferrostatin-1) to confirm mechanism.

• Mitochondrial Lipid Metabolism Assessment:

  • Isolate mitochondria and measure cardiolipin content and oxidation status.

  • Correlate with DNAJC15 expression levels determined by quantitative Western blot.

  • Analyze lipid droplet accumulation using BODIPY staining and microscopy.

  • Perform lipidomic analysis on cells with variable DNAJC15 expression.

• Chemotherapy Response Correlation:

  • Treat cells with cisplatin at various concentrations.

  • Measure DNAJC15 expression, lipid peroxidation, and cell viability simultaneously.

  • Use ferroptosis inhibitors to determine contribution to cell death mechanism.

  • Perform immunohistochemistry on patient samples to correlate DNAJC15 levels with treatment outcomes.

This multi-parameter approach can help elucidate how DNAJC15 enhances cancer cell vulnerability to chemotherapy-induced ferroptosis, as observed in ovarian cancer models where high DNAJC15 expression promoted sensitivity to cisplatin through ferroptosis activation .

How can researchers quantitatively assess the relationship between DNAJC15 expression and mitochondrial function?

To rigorously quantify the relationship between DNAJC15 expression and mitochondrial function, researchers should implement the following comprehensive protocol:

• Titrated Expression Analysis:

  • Generate cell lines with variable DNAJC15 expression (knockout, knockdown, wildtype, and overexpression).

  • Quantify DNAJC15 protein levels by Western blot using HRP-conjugated antibodies with standard curve calibration.

  • Plot expression levels against functional parameters to establish dose-response relationships.

• Mitochondrial Function Assessment Battery:

  • Respiratory chain activity: Measure oxygen consumption rate (OCR) using Seahorse XF analyzer with specific inhibitors to determine complex I-driven respiration.

  • Membrane potential: Quantify using TMRM or JC-1 dyes with flow cytometry or live-cell imaging.

  • ATP production: Utilize luciferase-based ATP assays normalized to mitochondrial content.

  • Supercomplex formation: Analyze using blue native PAGE followed by Western blotting.

• Data Integration and Normalization:

  • Normalize all parameters to mitochondrial mass (determined by mitochondrial protein content or mtDNA copy number).

  • Generate multiparameter correlation matrices.

  • Apply principal component analysis to identify key variables affected by DNAJC15 expression.

This methodological approach aligns with published findings that DNAJC15 negatively regulates respiratory chain activity, with its loss leading to increased complex I activity, enhanced mitochondrial membrane potential, and elevated ATP production . The quantitative correlation between expression levels and functional impacts can provide insight into the threshold effects and potential therapeutic windows for interventions targeting DNAJC15-dependent pathways.

What considerations should be made when using DNAJC15 antibodies for immunoprecipitation of mitochondrial protein complexes?

Immunoprecipitation (IP) of mitochondrial protein complexes containing DNAJC15 requires special considerations due to the membrane localization and complex formation characteristics:

• Mitochondrial Membrane Solubilization Strategy:

  • Evaluate multiple detergents: digitonin (0.5-2%) preserves supercomplexes, while n-dodecyl β-D-maltoside (0.5-1%) or Triton X-100 (0.5-1%) provide stronger solubilization.

  • Perform titration experiments to determine optimal detergent concentration for solubilizing DNAJC15 without disrupting relevant interactions.

  • Consider gradient solubilization approaches for sequential extraction of membrane proteins.

• IP Protocol Optimization:

  • Pre-clear lysates thoroughly to reduce non-specific binding.

  • Use unconjugated primary antibodies coupled to protein A/G beads rather than directly conjugated antibodies for flexible elution options.

  • Include appropriate controls: IgG control, input sample, and when possible, DNAJC15-knockout samples.

  • Consider crosslinking approaches for capturing transient interactions.

  • Perform IPs at 4°C overnight for maximum recovery of complexes.

• Post-IP Analysis Considerations:

  • For Western blot detection of co-immunoprecipitated proteins, choose denaturing conditions carefully to avoid antibody chain interference.

  • Consider on-bead digestion followed by mass spectrometry for unbiased complex composition analysis.

  • When probing for specific interactions (e.g., with complex I components), select antibodies against subunits physically accessible in the assembled complex.

These approaches have been successfully applied to demonstrate that DNAJC15 interacts preferentially with complex I of the electron transfer chain at the mitochondrial inner membrane . Researchers should be mindful that DNAJC15's role in regulating supercomplex formation may make some interactions detectable only under specific solubilization conditions that preserve these higher-order structures.

How does DNAJC15 expression influence metabolic adaptation in stress conditions?

DNAJC15's role in metabolic adaptation during stress conditions represents an emerging research area with significant implications for disease understanding. A comprehensive experimental approach should include:

• Stress-Specific Expression Profiling:

  • Subject cells/tissues to relevant stressors: nutrient deprivation, hypoxia, oxidative stress, and chemotherapeutic agents.

  • Quantify DNAJC15 protein levels using HRP-conjugated antibodies via Western blot or immunohistochemistry.

  • Assess subcellular localization changes under stress conditions using fractionation followed by immunoblotting.

  • Monitor real-time expression changes using reporter systems linked to the DNAJC15 promoter.

• Metabolic Flux Analysis Under Stress:

  • Perform stable isotope-resolved metabolomics (SIRM) using 13C-labeled glucose, glutamine, or fatty acids.

  • Compare metabolic flux distributions between DNAJC15-normal and DNAJC15-deficient systems under stress.

  • Focus on TCA cycle intermediates, lipid metabolism, and ATP production pathways.

  • Correlate with mitochondrial respiration measurements using Seahorse technology.

• Conditional Knockout Stress Response Assessment:

  • Generate inducible DNAJC15 knockout systems for temporal control.

  • Subject to metabolic stress conditions:

    • Fasting (to model findings on hepatic lipid accumulation)

    • High-cholesterol diet challenges

    • Chemotherapeutic treatment (particularly cisplatin)

  • Measure stress adaptation markers: lipid peroxidation, ATP/ADP ratio, NADH/NAD+ ratio, and ROS levels.

  • Assess ferroptosis susceptibility markers in cancer models.

Research has shown that DNAJC15 deficiency can mitigate pathological consequences of metabolic alterations, leading to a "rapid metabolism" phenotype that prevents lipid accumulation in liver during fasting or high-cholesterol diets . Conversely, high DNAJC15 expression in cancer contexts appears to promote vulnerability to ferroptosis, potentially explaining chemotherapy sensitivity in certain tumors . These findings suggest that DNAJC15 may serve as a metabolic checkpoint that determines cellular resilience to specific stressors.

How can signal-to-noise ratio be optimized when using HRP-conjugated DNAJC15 antibodies?

Optimizing signal-to-noise ratio is critical for accurate DNAJC15 detection, particularly in tissues with variable expression levels. The following technical refinements can significantly improve results:

• Blocking Optimization Protocol:

  • Compare protein-based blockers (BSA, milk) with synthetic blockers.

  • For Western blots: 5% non-fat milk typically provides good blocking, but switch to 3-5% BSA if phospho-specific detection is needed.

  • For IHC applications: Consider species-matched serum (1-10%) or commercial blocking solutions.

  • Perform sequential blocking: first with protein blocker, then with 0.1-0.3% Tween-20 to reduce hydrophobic interactions.

• Antibody Concentration Titration:

  • Perform systematic dilution series (1:500, 1:1000, 1:2000, 1:4000) to identify optimal concentration .

  • Evaluate signal-to-noise ratio at each concentration using positive and negative controls.

  • Consider extended incubation (overnight at 4°C) with more dilute antibody to improve specificity.

• Detection System Enhancement:

  • For Western blots: Use enhanced chemiluminescence (ECL) substrates calibrated to the expected expression level.

  • For low abundance targets: Consider signal amplification systems compatible with HRP (tyramide signal amplification).

  • Optimize exposure times through multiple short exposures rather than a single long exposure.

• Sample-Specific Considerations:

  • For brain tissue: Add 0.1% Triton X-100 to antibody diluent to improve penetration.

  • For liver samples: Consider additional washing steps to remove endogenous biotin.

  • For cell lines with low expression: Concentrate samples through mitochondrial isolation protocols.

These optimization strategies can help achieve the high sensitivity needed for detecting DNAJC15's native expression while maintaining specificity. Validated protocols have shown that DNAJC15 can be reliably detected in tissues such as mouse liver, human brain, and human liver when appropriate optimization steps are taken .

What strategies can improve detection of DNAJC15 in fixed tissue samples?

Detection of DNAJC15 in fixed tissue samples presents unique challenges due to its mitochondrial localization and potential epitope masking during fixation. The following comprehensive approach can significantly enhance detection sensitivity and specificity:

• Antigen Retrieval Optimization:

  • Primary recommendation: Use TE buffer at pH 9.0 for heat-induced epitope retrieval .

  • Alternative approach: Citrate buffer at pH 6.0 may be effective for certain tissue types .

  • Perform retrieval in a pressure cooker (20 minutes) rather than microwave for more consistent results.

  • Consider a dual retrieval approach: proteolytic enzyme treatment (5-10 minutes) followed by heat-induced retrieval.

• Signal Enhancement Protocol:

  • Implement tyramide signal amplification (TSA) system compatible with HRP-conjugated antibodies.

  • Use avidin-biotin complex (ABC) method if working with biotinylated primary antibodies.

  • Consider polymer detection systems for improved sensitivity and reduced background.

  • Optimize chromogen development time through timed monitoring.

• Background Reduction Techniques:

  • Block endogenous peroxidase with 0.3% H₂O₂ in methanol (10 minutes) prior to antibody incubation.

  • Include avidin-biotin blocking step for tissues with high endogenous biotin (liver, kidney).

  • Add 0.1-0.3% Triton X-100 to antibody diluent for improved penetration and reduced non-specific binding.

  • Consider mouse-on-mouse blocking for mouse tissues when using mouse-derived antibodies.

• Incubation Optimization:

  • Extended primary antibody incubation (overnight at 4°C) at dilutions between 1:50-1:500 .

  • Use humidity chambers to prevent section drying.

  • Consider oscillating platform for gentle agitation during incubation.

  • Optimize washing protocols: 3 x 5-minute washes with 0.1% Tween-20 in PBS.

When implementing these approaches, researchers should validate results using known positive controls such as human liver tissue, which has been documented to show reliable DNAJC15 expression in immunohistochemical applications . The mitochondrial localization pattern should appear as punctate cytoplasmic staining, distinct from nuclear or diffuse cytoplasmic patterns that might indicate non-specific binding.

How can multiplex detection systems be employed to study DNAJC15 in relation to other mitochondrial proteins?

Multiplex detection systems offer powerful approaches for investigating DNAJC15's relationship with other mitochondrial proteins in the same sample. The following methodological strategies enable comprehensive analysis of protein interactions and co-localization:

• Sequential Fluorescent Western Blotting:

  • Strip and reprobe technique:

    • Perform initial detection with HRP-conjugated DNAJC15 antibody.

    • Document results thoroughly.

    • Strip membrane with commercial stripping buffer (optimize time to ensure complete removal).

    • Reprobe with antibodies against mitochondrial markers (VDAC, COX IV) or interacting partners (complex I components).

  • Multiplexed detection using antibodies from different species:

    • Use HRP-conjugated DNAJC15 antibody with fluorescently-labeled secondary antibodies for other targets.

    • Employ narrow-spectrum fluorophores to minimize spectral overlap.

    • Include appropriate controls to confirm specificity of each detection channel.

• Multiplex Immunohistochemistry Protocol:

  • Tyramide-based multiplex IHC:

    • Perform sequential rounds of antibody staining using HRP-conjugated antibodies.

    • Apply tyramide-fluorophores for signal amplification and covalent binding.

    • Between rounds, perform complete HRP inactivation with hydrogen peroxide.

    • This approach allows detection of 5-7 proteins on a single tissue section.

  • Chromogenic multiplex IHC:

    • Use different chromogens (DAB, AEC, Fast Red) for distinct visualization of each target.

    • Optimize sequence of antibody application based on epitope abundance.

• Proximity-Based Detection Methods:

  • Proximity ligation assay (PLA):

    • Apply DNAJC15 antibody alongside antibodies against potential interacting partners.

    • Use species-specific PLA probes followed by rolling circle amplification.

    • Positive signals appear as fluorescent spots only where proteins are in close proximity (<40 nm).

  • FRET/FLIM analysis:

    • Label DNAJC15 and interacting partners with appropriate FRET donor/acceptor pairs.

    • Measure energy transfer as indicator of protein-protein interaction.

• Mass Cytometry (CyTOF) Application:

  • Label antibodies with distinct metal isotopes.

  • Perform staining on single-cell suspensions from tissues or cell cultures.

  • Analyze using mass cytometry for simultaneous detection of 30+ proteins.

  • Particularly valuable for heterogeneous samples with variable DNAJC15 expression.

These approaches allow researchers to investigate DNAJC15's relationship with other mitochondrial proteins, particularly its preferential interaction with complex I of the electron transfer chain and its role in regulating supercomplex formation . When analyzing results, focus on co-localization patterns, changes in relative expression under different conditions, and potential competitive or cooperative binding relationships between DNAJC15 and other mitochondrial proteins.

What approaches can verify antibody specificity for DNAJC15 in experimental systems?

Establishing and verifying antibody specificity is crucial for reliable DNAJC15 research. The following comprehensive validation approach ensures confidence in experimental results:

• Genetic Validation Strategy:

  • CRISPR/Cas9 knockout controls:

    • Generate DNAJC15 knockout cell lines.

    • Compare Western blot results between wildtype and knockout samples.

    • The specific band should be absent in knockout samples.

  • siRNA/shRNA knockdown validation:

    • Perform titrated knockdown with increasing siRNA concentrations.

    • Demonstrate proportional reduction in the target band intensity.

    • Include non-targeting siRNA controls to rule out off-target effects.

  • Overexpression verification:

    • Express tagged DNAJC15 constructs (His, FLAG, GFP).

    • Confirm detection of overexpressed protein at expected molecular weight plus tag size.

    • Compare detection using both tag-specific and DNAJC15-specific antibodies.

• Immunological Validation Methods:

  • Peptide competition assay:

    • Pre-incubate antibody with excess immunizing peptide (DNAJC15 amino acids 70-150) .

    • Specific signals should be abolished or significantly reduced.

    • Non-specific signals will remain unaffected.

  • Independent antibody validation:

    • Compare results using antibodies targeting different DNAJC15 epitopes.

    • Concordant results with multiple antibodies suggest specific detection.

    • Consider both polyclonal and monoclonal antibodies in comparison.

• Biochemical Verification Techniques:

  • Subcellular fractionation analysis:

    • Prepare nuclear, cytoplasmic, and mitochondrial fractions.

    • DNAJC15 should be enriched in mitochondrial fractions .

    • Verify with established compartment markers (e.g., VDAC for mitochondria).

  • Mass spectrometry confirmation:

    • Immunoprecipitate using DNAJC15 antibody.

    • Analyze by mass spectrometry to confirm identity.

    • Search for peptides matching the DNAJC15 sequence.

• Species Reactivity Verification:

  • Test antibody against samples from multiple species when cross-reactivity is claimed.

  • Verify reactivity with human, mouse, and rat samples if working with antibodies claiming multi-species detection .

  • Consider species-specific positive controls: human brain tissue, mouse liver tissue, rat testis .

These validation approaches align with published research on DNAJC15, which has demonstrated specific detection at 18-20 kDa in Western blot applications and distinct mitochondrial localization patterns in immunohistochemistry . Implementing multiple validation strategies provides the highest confidence in antibody specificity and experimental results.

What methodological approaches can optimize DNAJC15 antibody use in challenging samples like brain tissue?

Brain tissue presents unique challenges for protein detection due to its complex cellular composition, high lipid content, and variable protein expression levels. The following specialized approaches can optimize DNAJC15 detection in neural tissues:

• Tissue Preparation and Fixation Protocol:

  • For fresh tissues:

    • Rapid post-mortem collection (<30 minutes) to prevent protein degradation.

    • Micro-dissection of relevant brain regions with established DNAJC15 expression.

    • Snap freezing in liquid nitrogen with pre-chilled isopentane to prevent ice crystal formation.

  • For fixed tissues:

    • Short fixation (24-48 hours) in 4% paraformaldehyde.

    • Graduated alcohol dehydration to prevent tissue distortion.

    • Low-temperature paraffin embedding to minimize protein denaturation.

• Enhanced Antigen Retrieval Methods:

  • Two-step antigen retrieval:

    • Initial enzymatic treatment with proteinase K (1-5 μg/ml, 5 minutes).

    • Follow with heat-induced epitope retrieval in TE buffer pH 9.0 at 97°C for 20 minutes .

  • pH gradient analysis:

    • Test retrieval buffers across pH range (6.0, 7.0, 8.0, 9.0).

    • Document signal intensity and background at each pH value.

    • Optimize time-temperature combinations for selected pH.

• Signal Amplification Cascade:

  • Catalyzed signal amplification system:

    • Utilize biotinylated tyramide for signal deposition.

    • Apply streptavidin-HRP secondary detection.

    • This provides 10-50× signal enhancement compared to standard detection.

  • Quantum dot conjugation:

    • Consider quantum dot-conjugated secondary antibodies for improved sensitivity and stability.

    • Provides photostable signal for extended imaging sessions.

    • Enables multiplex detection with minimal spectral overlap.

• Background Reduction Strategies for Neural Tissue:

  • Lipofuscin autofluorescence reduction:

    • Pre-treat sections with 0.1% Sudan Black B in 70% ethanol for 20 minutes.

    • Alternatively, use TrueBlack® lipofuscin autofluorescence quencher.

  • Specialized blocking:

    • Mouse Brain Slice Blocking Buffer (2% BSA, 0.2% milk, 0.1% gelatin, 0.05% saponin in PBS).

    • Add 0.1-0.3% Triton X-100 for improved antibody penetration.

    • Include 10% normal serum matching secondary antibody species.

• Optimized Detection Parameter:

  • For immunohistochemistry:

    • Extend primary antibody incubation to 48-72 hours at 4°C with gentle agitation.

    • Use thin sections (5-7 μm) to improve antibody penetration.

    • Consider free-floating section technique for improved antibody access.

  • For Western blot:

    • Include phosphatase inhibitors in lysis buffer to preserve post-translational modifications.

    • Consider gradient gels (4-15%) for optimal protein separation.

    • Increase protein loading (50-80 μg) for regions with lower DNAJC15 expression.

These specialized approaches have been successful in detecting DNAJC15 in brain tissue, which serves as a validated positive control for antibody performance . The optimization of these parameters allows researchers to investigate DNAJC15's role in neurological contexts, including potential involvement in neurodegenerative disorders linked to mitochondrial dysfunction.

How can DNAJC15 antibodies be utilized to study its role in cancer chemoresistance mechanisms?

Recent research has established important connections between DNAJC15 expression and cancer chemotherapy response, particularly in ovarian cancer. The following methodological approach allows comprehensive investigation of this relationship:

• Clinical Sample Analysis Protocol:

  • Tissue microarray construction:

    • Prepare arrays containing matched pre- and post-chemotherapy tumor samples.

    • Include chemotherapy-responsive and resistant cases.

    • Perform immunohistochemistry with optimized DNAJC15 antibody dilutions (1:50-1:500) .

    • Quantify expression using digital pathology scoring systems.

  • Correlation analysis:

    • Match DNAJC15 expression with treatment response data.

    • Perform Kaplan-Meier survival analysis based on expression levels.

    • Conduct multivariate analysis to control for confounding clinical factors.

• In Vitro Resistance Model Characterization:

  • Paired sensitive/resistant cell line panel:

    • Establish cisplatin-resistant cell lines through incremental drug exposure.

    • Quantify DNAJC15 expression by Western blot with HRP-conjugated antibodies.

    • Correlate expression with IC50 values for relevant chemotherapeutics.

  • DNAJC15 modulation:

    • Create overexpression and knockdown/knockout models.

    • Assess changes in drug sensitivity through dose-response assays.

    • Determine mechanism through ferroptosis markers and mitochondrial function assays.

• Ferroptosis Mechanism Investigation:

  • Lipid peroxidation assessment:

    • Measure lipid peroxidation using C11-BODIPY in cells with variable DNAJC15 expression.

    • Quantify lipid droplet accumulation using BODIPY staining.

    • Test ferroptosis inhibitors (Ferrostatin-1) to confirm mechanism.

  • Mitochondrial function correlation:

    • Measure mitochondrial membrane potential with TMRM or JC-1 dyes.

    • Assess ROS production using MitoSOX.

    • Correlate with DNAJC15 expression levels determined by immunoblotting.

• Therapeutic Response Prediction:

  • Develop immunohistochemistry-based scoring system:

    • Establish DNAJC15 expression thresholds associated with treatment response.

    • Validate in independent patient cohorts.

    • Integrate with existing biomarkers for improved prediction.

  • Combination therapy approach:

    • Test DNAJC15 modulators with conventional chemotherapeutics.

    • Assess sensitivity to ferroptosis inducers based on DNAJC15 status.

    • Evaluate potential for targeted therapy based on expression levels.

This methodological framework addresses the finding that high levels of DNAJC15 are associated with increased sensitivity to cisplatin in ovarian cancer cells through ferroptosis promotion . By systematically investigating DNAJC15's role in chemoresistance, researchers can potentially develop predictive biomarkers and therapeutic approaches targeting this pathway.

What methodological considerations are important when studying DNAJC15 in metabolic disorders using antibody-based approaches?

Investigating DNAJC15's role in metabolic disorders requires specialized methodological approaches that address its function in mitochondrial respiration and lipid metabolism. The following comprehensive protocol provides guidance for such studies:

• Animal Model Tissue Analysis:

  • Diet-induced metabolic challenge:

    • Subject animals to fasting or high-cholesterol diet challenges .

    • Harvest tissues (liver, muscle, adipose) at defined timepoints.

    • Process for both protein expression (Western blot) and histological analysis (IHC).

  • Standardized tissue collection protocol:

    • Collect tissues at consistent circadian timepoints to control for metabolic fluctuations.

    • Process immediately to preserve post-translational modifications.

    • Prepare both fresh-frozen samples for biochemical analysis and fixed samples for morphological studies.

• Subcellular Fractionation and Analysis:

  • Mitochondrial isolation:

    • Implement density gradient centrifugation for high-purity mitochondrial fractions.

    • Further subfractionate into outer membrane, inner membrane, and matrix components.

    • Analyze DNAJC15 distribution using Western blot with HRP-conjugated antibodies.

  • Respiratory complex association:

    • Solubilize mitochondria with digitonin to preserve supercomplexes.

    • Perform blue native PAGE to separate intact complexes.

    • Identify DNAJC15 association with respiratory complexes through Western blot.

• Metabolic Function Correlation:

  • Respirometry analysis:

    • Measure oxygen consumption in response to metabolic challenges.

    • Correlate with DNAJC15 expression levels in the same samples.

    • Assess sensitivity to complex I-specific inhibitors (rotenone).

  • Lipid metabolism assessment:

    • Quantify lipid accumulation using Oil Red O staining.

    • Measure triglyceride and cholesterol content biochemically.

    • Correlate with DNAJC15 expression and localization.

• Tissue-Specific Expression Patterns:

  • Multi-tissue Western blot panel:

    • Analyze DNAJC15 expression across metabolically active tissues.

    • Use tissue-specific positive controls (liver, brain, muscle) .

    • Quantify relative expression levels normalized to mitochondrial markers.

  • Immunohistochemical profiling:

    • Optimize antigen retrieval methods for different tissues.

    • Quantify cellular and subcellular distribution patterns.

    • Correlate with metabolic stress markers and disease progression.

This methodological approach addresses findings that DNAJC15 deficiency creates a "rapid metabolism" phenotype that can prevent pathological lipid accumulation in response to metabolic stressors such as fasting and high-cholesterol diets . By systematically analyzing DNAJC15's expression and function in metabolic contexts, researchers can better understand its potential as a therapeutic target in metabolic disorders.

How can researchers quantitatively assess DNAJC15 expression changes in response to cellular stress conditions?

Quantitative assessment of DNAJC15 expression changes under stress conditions requires rigorous methodological approaches that account for various confounding factors. The following comprehensive protocol enables precise measurement across different experimental systems:

• Standardized Stress Induction Protocol:

  • Oxidative stress:

    • Apply hydrogen peroxide (50-500 μM) or paraquat (10-100 μM) in dose-response and time-course experiments.

    • Confirm stress induction through ROS measurement (DCF-DA assay) and viability assessment.

  • Metabolic stress:

    • Implement glucose deprivation, serum starvation, or hypoxia (1-5% O₂).

    • Monitor metabolic adaptations through lactate production and ATP/ADP ratio measurement.

  • Pharmacological stress:

    • Apply cisplatin (1-10 μM) or other chemotherapeutics with established ferroptosis-inducing potential .

    • Confirm stress response through appropriate cellular markers.

• Multi-parameter Expression Analysis:

  • Quantitative Western blot:

    • Include recombinant DNAJC15 protein standards for absolute quantification.

    • Apply HRP-conjugated DNAJC15 antibodies at optimized dilutions (1:1000-1:4000) .

    • Implement digital image analysis with lane normalization.

    • Normalize to both total protein (Ponceau S) and mitochondrial markers (VDAC, COX IV).

  • Real-time expression monitoring:

    • Generate reporter cell lines with DNAJC15 promoter driving luciferase expression.

    • Perform time-lapse luminescence imaging during stress induction.

    • Correlate with protein levels measured by immunoblotting.

• Single-Cell Analysis Approach:

  • Flow cytometry protocol:

    • Fix and permeabilize cells with optimized buffers for mitochondrial protein detection.

    • Apply directly conjugated DNAJC15 antibodies at validated concentrations.

    • Co-stain for stress markers and cell type identifiers.

    • Analyze population distributions rather than just mean values.

  • Imaging cytometry:

    • Combine flow cytometry with microscopy for morphological correlation.

    • Quantify subcellular distribution changes under stress.

    • Measure co-localization with mitochondrial markers and stress granules.

• Comprehensive Data Integration:

  • Multi-omics correlation:

    • Integrate protein expression data with transcriptomics (RT-qPCR or RNA-seq).

    • Assess post-transcriptional regulation through RNA/protein ratio analysis.

    • Correlate with metabolomics data focusing on mitochondrial metabolites.

  • Systems biology approach:

    • Develop mathematical models of DNAJC15 expression dynamics.

    • Incorporate feedback loops with stress response pathways.

    • Validate model predictions with targeted experiments.

This quantitative framework allows researchers to precisely characterize how DNAJC15 expression changes in response to various stressors, building on findings that its expression levels correlate with distinct cellular phenotypes in contexts such as cancer chemoresistance and metabolic adaptation . The ability to quantitatively track these changes is essential for understanding DNAJC15's role as a potential biomarker or therapeutic target in stress-related pathologies.

What emerging technologies could enhance DNAJC15 protein interaction studies beyond current antibody capabilities?

While antibody-based approaches have been instrumental in DNAJC15 research, emerging technologies offer new possibilities for studying its interactions and functions with unprecedented resolution and throughput:

• Proximity-Based Proteomic Approaches:

  • BioID/TurboID methodology:

    • Generate DNAJC15-BioID fusion constructs for expression in relevant cell types.

    • Allow biotin ligase to biotinylate proximal proteins in living cells.

    • Purify biotinylated proteins and identify by mass spectrometry.

    • This approach captures transient and weak interactions missed by traditional co-IP.

  • APEX2 proximity labeling:

    • Express DNAJC15-APEX2 fusion in mitochondria.

    • Add biotin-phenol substrate and H₂O₂ for rapid (1 minute) labeling of nearby proteins.

    • Particularly valuable for mapping the DNAJC15 interaction landscape within the mitochondrial inner membrane.

• Advanced Structural Biology Techniques:

  • Cryo-electron microscopy:

    • Purify native complexes containing DNAJC15 through gentle extraction methods.

    • Determine high-resolution structures of DNAJC15 in complex with respiratory chain components.

    • Map interaction interfaces to guide therapeutic development.

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

    • Probe conformational dynamics of DNAJC15 alone and in complex with partners.

    • Map binding interfaces through differential solvent exposure.

    • Understand allosteric effects of binding events.

• Live-Cell Interaction Monitoring:

  • CRISPR knock-in fluorescent tagging:

    • Generate endogenous DNAJC15 tagged with fluorescent proteins.

    • Combine with tagged interaction partners for real-time FRET analysis.

    • Monitor interactions under various metabolic conditions and stressors.

  • Split protein complementation assays:

    • Develop DNAJC15 constructs with split luciferase or fluorescent protein fragments.

    • When interaction occurs, functional reporter protein is reconstituted.

    • Allows high-throughput screening of conditions affecting interactions.

• Next-Generation Functional Genomics:

  • CRISPR interference/activation screens:

    • Apply CRISPRi/CRISPRa libraries targeting genes potentially related to DNAJC15 function.

    • Measure effects on DNAJC15-dependent phenotypes (e.g., chemosensitivity, metabolic parameters).

    • Identify novel regulatory pathways and functional connections.

  • Domain-focused mutagenesis:

    • Generate comprehensive libraries of DNAJC15 variants with mutations across the protein.

    • Assess functional consequences on interaction with complex I and other partners.

    • Map critical functional domains with single-amino acid resolution.

These emerging technologies can help resolve current knowledge gaps, particularly regarding how DNAJC15 preferentially interacts with complex I of the electron transfer chain and how it impairs the formation of respiratory supercomplexes . They may also provide insight into DNAJC15's role in promoting ferroptosis and chemotherapy sensitivity in cancer contexts , potentially leading to novel therapeutic approaches targeting these pathways.

How might quantitative proteomics complement antibody-based approaches for DNAJC15 research in complex disease models?

Quantitative proteomics offers powerful complementary approaches to antibody-based detection for DNAJC15 research, particularly in complex disease contexts where comprehensive protein network analysis is essential:

• Global Proteomic Profiling Strategy:

  • SWATH-MS (Sequential Window Acquisition of all Theoretical Mass Spectra):

    • Generate comprehensive digital proteome maps of samples with variable DNAJC15 expression.

    • Quantify thousands of proteins simultaneously without antibody limitations.

    • Identify co-regulated protein networks through correlation analysis.

    • Apply to tissues from metabolic disease models and cancer samples with varying DNAJC15 levels.

  • TMT/iTRAQ multiplexed quantification:

    • Compare up to 16 samples in a single experiment through isobaric labeling.

    • Ideal for time-course studies of DNAJC15 regulation during stress response.

    • Provides statistical power through biological and technical replicates.

• Post-Translational Modification Mapping:

  • Phosphoproteomics:

    • Enrich for phosphopeptides using titanium dioxide or IMAC.

    • Identify phosphorylation sites on DNAJC15 and changes in response to stimuli.

    • Map signaling networks affected by DNAJC15 expression.

  • Comprehensive PTM analysis:

    • Identify acetylation, ubiquitination, SUMOylation patterns on DNAJC15.

    • Correlate modifications with functional states and protein stability.

    • Discover regulatory mechanisms controlling DNAJC15 activity.

• Targeted Proteomics Approach:

  • Parallel Reaction Monitoring (PRM):

    • Develop targeted assays for absolute quantification of DNAJC15 and interacting partners.

    • Achieve higher sensitivity than global approaches for low-abundance proteins.

    • Validate findings from antibody-based studies with orthogonal methodology.

  • Targeted PTM quantification:

    • Monitor specific modified forms of DNAJC15 with high sensitivity.

    • Track changes in modification state during disease progression.

    • Correlate with functional outcomes in relevant models.

• Protein Interactome Analysis:

  • Proximity labeling combined with quantitative proteomics:

    • Express DNAJC15-BioID in disease models and controls.

    • Identify differential interactors through quantitative comparison.

    • Discover context-specific interactions relevant to disease mechanisms.

  • Cross-linking mass spectrometry (XL-MS):

    • Capture interaction networks through chemical cross-linking.

    • Map protein-protein interfaces at amino acid resolution.

    • Identify structural changes in complexes under disease conditions.