DNAJB9 Antibody

What is DNAJB9 and why is it significant in research?

DNAJB9 (DnaJ Heat Shock Protein Family Hsp40 Member B9) is a protein encoded by the DNAJB9 gene in humans. It has gained significant research importance as it has been identified as a specific biomarker for Fibrillary Glomerulonephritis (FGN). DNAJB9 is a co-chaperone for Hsp70 protein HSPA5/BiP that functions as a key repressor of the ERN1/IRE1-mediated unfolded protein response (UPR). It is localized to the endoplasmic reticulum and plays a role in endoplasmic reticulum-associated degradation (ERAD) of misfolded proteins . The protein is approximately 25.5 kilodaltons in mass and is also known by several aliases including ERdj4, MDG-1, MDG1, and MST049 .

How specific is DNAJB9 as a biomarker for Fibrillary Glomerulonephritis?

DNAJB9 has demonstrated exceptional specificity as a biomarker for Fibrillary Glomerulonephritis (FGN). Research has shown that DNAJB9 has 98% sensitivity and >99% specificity for FGN diagnosis . In another study, DNAJB9 was found to be an FGN marker with 100% sensitivity and 100% specificity, making it a powerful diagnostic tool . It was present in all FGN samples tested but was not detectable in normal tissue, amyloidosis samples, or non-FGN glomerular disease samples. The magnitude and specificity of DNAJB9 overabundance in FGN also suggests that this protein has a role in FGN pathogenesis .

What are the common applications of DNAJB9 antibodies in research?

DNAJB9 antibodies are utilized across multiple research applications:

Research has demonstrated these applications in multiple publications focusing on DNAJB9's role in FGN diagnosis and protein characterization .

What is the optimal protocol for DNAJB9 immunohistochemistry in FGN diagnosis?

For optimal DNAJB9 immunohistochemistry in FGN diagnosis, researchers should follow these methodological steps:

• Tissue Preparation: Use formalin-fixed, paraffin-embedded (FFPE) kidney biopsy specimens.

• Antigen Retrieval: Use TE buffer at pH 9.0 as the preferred method, although citrate buffer at pH 6.0 can serve as an alternative .

• Antibody Selection: Use validated anti-DNAJB9 antibodies at the appropriate dilution (typically 1:200-1:500 for IHC) .

• Detection System: Employ a standard indirect immunoperoxidase technique with appropriate controls.

• Interpretation: Look for strong, homogeneous, smudgy DNAJB9 staining of glomerular deposits, which is characteristic of FGN. This pattern is distinct from the granular cytoplasmic staining observed in normal cells .

Research has demonstrated that proper DNAJB9 immunohistochemistry can replace the need for electron microscopy in many cases of suspected FGN, streamlining the diagnostic process .

How should researchers validate DNAJB9 antibody specificity for experimental use?

Validating DNAJB9 antibody specificity is crucial for reliable experimental results. A comprehensive validation approach should include:

• Western Blot Analysis: Confirm a single band at the expected molecular weight (26-30 kDa for DNAJB9) .

• Knockout/Knockdown Controls: Test antibody on DNAJB9 knockout or knockdown samples to confirm specificity.

• Multiple Antibody Comparison: Use at least two different antibodies targeting different epitopes of DNAJB9.

• Cross-reactivity Testing: Evaluate potential cross-reactivity with other DNAJ family members, particularly close paralogs like DNAJB4 .

• Immunoprecipitation Followed by Mass Spectrometry: Confirm that the immunoprecipitated protein is indeed DNAJB9.

• Tissue Panel Testing: Verify expected expression patterns across multiple tissues (as shown in Table 2 in reference ), where DNAJB9 should show granular cytoplasmic staining in various cell types.

Proper validation ensures that experimental findings genuinely reflect DNAJB9 biology rather than antibody artifacts.

What are the recommended storage conditions and handling procedures for DNAJB9 antibodies?

For optimal DNAJB9 antibody performance and longevity:

• Storage Temperature: Store at -20°C. Most commercial DNAJB9 antibodies remain stable for one year after shipment when stored properly .

• Buffer Composition: Most DNAJB9 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Aliquoting: For larger volumes, aliquoting is recommended to avoid repeated freeze-thaw cycles, although some manufacturers indicate it may be unnecessary for -20°C storage of small volumes (e.g., 20μL) .

• Working Solution: Dilute antibodies immediately before use in appropriate buffer.

• Handling: Avoid contamination and maintain sterile conditions when handling antibody solutions.

• Shipping: Antibodies are typically shipped on wet ice and should be stored immediately upon receipt .

Following these guidelines will help maintain antibody efficacy and experimental reproducibility.

How can DNAJB9 immunohistochemistry differentiate FGN from amyloidosis and other mimicking conditions?

DNAJB9 immunohistochemistry provides a powerful tool to differentiate FGN from conditions with similar histological appearances:

• Pattern Recognition: FGN shows strong, homogeneous, smudgy DNAJB9 staining in glomerular deposits, while amyloidosis and other non-FGN glomerular diseases are consistently negative for DNAJB9 .

• Co-staining Approach: Dual immunofluorescence staining for DNAJB9 and Ig-γ demonstrates co-localization in FGN cases, showing that DNAJB9 is deposited with immune complexes. This pattern is not seen in amyloidosis or other conditions .

• Complementary Tests: While Congo red negativity is typically used to exclude amyloidosis, DNAJB9 positivity provides positive confirmation of FGN, increasing diagnostic certainty.

• Sensitivity and Specificity: Studies have shown that DNAJB9 immunohistochemistry has 98-100% sensitivity and >99% specificity for FGN diagnosis, making it more reliable than morphological assessment alone .

• Challenging Cases: In cases with atypical features, DNAJB9 staining can be definitive. For instance, the research noted that even unusual FGN cases (such as those with only IgG staining without κ or λ light chains) may lack DNAJB9 staining, helping to identify variant forms of the disease .

This approach has transformed the diagnostic algorithm for FGN, potentially eliminating the need for electron microscopy in many cases.

Can DNAJB9 antibodies be used for monitoring disease progression or recurrence after kidney transplantation?

DNAJB9 antibodies have demonstrated value in monitoring disease progression and recurrence:

• Transplant Monitoring: Research has shown that DNAJB9 immunohistochemistry can detect FGN recurrence in kidney transplant recipients, allowing for earlier intervention .

• Sequential Biopsy Assessment: By performing DNAJB9 staining on sequential biopsies, researchers can track the deposition pattern and intensity over time, potentially correlating with disease activity.

• Methodological Approach: For transplant monitoring, the same immunohistochemistry protocols used for initial diagnosis can be applied, with careful comparison to baseline patterns.

• Interpretation Challenges: In the transplant setting, interpretation may be complicated by rejection-related changes, requiring experienced pathologists to distinguish FGN recurrence from other pathologies.

• Research Applications: Beyond clinical monitoring, this approach provides insights into disease pathogenesis by enabling study of FGN progression and response to treatments in the transplanted kidney.

This application of DNAJB9 antibodies represents an important advance in post-transplant management of FGN patients.

What is the relationship between DNAJB9 expression and the unfolded protein response (UPR) in diseased tissues?

The relationship between DNAJB9 expression and the unfolded protein response is complex and context-dependent:

These findings highlight the unique nature of DNAJB9 accumulation in FGN and suggest distinct mechanisms from classical UPR activation.

What are the structural characteristics of DNAJB9 protein and how do they influence antibody epitope selection?

DNAJB9 protein structure has several features that influence antibody development and epitope selection:

• Domain Organization: DNAJB9 contains a J domain that is characteristic of the DNAJ/HSP40 family, which is critical for stimulating the ATPase activity of HSP70 proteins. Antibodies targeting this region should be carefully validated for specificity against other J domain-containing proteins .

• Sequence Coverage: Mass spectrometry studies have detected peptides distributed across the entire length of the mature DNAJB9 protein in FGN deposits, indicating that full-length protein (not truncated forms) accumulates in disease .

• Epitope Accessibility: In normal cells, DNAJB9 shows granular cytoplasmic staining consistent with ER localization. In FGN, the protein forms extracellular deposits with a distinctive smudgy pattern. Antibodies must recognize epitopes that remain accessible in both contexts .

• Conservation Across Species: DNAJB9 shows high conservation across species, with orthologs in canine, porcine, monkey, mouse, and rat models, allowing for cross-species applications of some antibodies .

• Immunogen Selection: Commercial antibodies use various strategies, including:

  • N-terminal region targeting (ARP46885_P050)

  • C-terminal targeting (C2C3 antibody)

  • Mid-region peptide targeting (aa 50-100 or aa 50-150)

For research applications, selecting antibodies targeting different epitopes can provide complementary data and validation.

What is the current understanding of DNAJB9 genetic variations and their potential role in FGN pathogenesis?

Current research on DNAJB9 genetic variations and FGN pathogenesis presents a complex picture:

These findings suggest that FGN pathogenesis likely involves post-transcriptional or post-translational mechanisms affecting DNAJB9 processing, degradation, or deposition rather than primary genetic alterations.

How do immunoelectron microscopy techniques enhance our understanding of DNAJB9 localization in FGN?

Immunoelectron microscopy (immuno-EM) has provided crucial insights into DNAJB9 localization in FGN:

• Fibril Association: Immuno-EM studies have directly demonstrated that DNAJB9 is localized to FGN fibrils themselves, not just to the surrounding matrix or cells. This provides strong evidence for DNAJB9 as an integral component of the pathological fibrils .

• Specificity of Association: Comparative immuno-EM studies on amyloid fibrils and immunotactoid glomerulopathy microtubules showed no DNAJB9 localization to these structures, confirming the specificity of DNAJB9 for FGN fibrils .

• Methodological Approach: The technique employs anti-DNAJB9 rabbit polyclonal antibody as the primary antibody and gold-conjugated goat anti-rabbit IgG as the secondary antibody, allowing direct visualization of DNAJB9 at the ultrastructural level .

• Control Measures: Proper controls (omitting the primary antibody) have demonstrated the absence of nonspecific staining with the gold-conjugated secondary antibody, validating the specificity of the observed patterns .

• Correlation with Light Microscopy: The immuno-EM findings correlate with light microscopy observations of DNAJB9 and IgG co-localization, creating a comprehensive picture of protein distribution across different scales of tissue organization .

These techniques have been instrumental in establishing DNAJB9 as not merely associated with but structurally integrated into FGN fibrils, fundamentally advancing our understanding of disease pathophysiology.

What are common challenges in DNAJB9 immunohistochemistry and how can they be addressed?

Researchers may encounter several challenges when performing DNAJB9 immunohistochemistry:

• Background Staining:

  • Challenge: Non-specific background can obscure specific DNAJB9 signals.

  • Solution: Optimize blocking steps using BSA or serum matching the secondary antibody host; consider using specialized blocking reagents for endogenous peroxidase and biotin.

• Antigen Retrieval Optimization:

  • Challenge: Inadequate epitope exposure can reduce sensitivity.

  • Solution: Compare TE buffer (pH 9.0) with citrate buffer (pH 6.0); optimize retrieval time and temperature for each tissue type .

• Distinguishing Normal vs. Pathological Staining:

  • Challenge: DNAJB9 shows normal granular cytoplasmic staining in various cell types.

  • Solution: Become familiar with normal staining patterns in tissues (Table 2 in reference ); pathological FGN deposits show distinctive homogeneous, smudgy extracellular patterns.

• Tissue Fixation Variables:

  • Challenge: Overfixation or underfixation can affect epitope availability.

  • Solution: Standardize fixation protocols; for archival tissues with variable fixation, extend antigen retrieval time.

• Antibody Selection:

  • Challenge: Different antibodies target different epitopes with varying sensitivities.

  • Solution: Compare multiple antibodies and optimize dilutions for each; consider using C-terminal antibodies for FGN diagnosis based on published validation .

Careful optimization and standardization of these variables will improve reproducibility and diagnostic accuracy.

How can researchers troubleshoot inconsistent Western blot results with DNAJB9 antibodies?

For consistent Western blot results with DNAJB9 antibodies:

• Sample Preparation Issues:

  • Challenge: Inconsistent protein extraction from different tissues.

  • Solution: Use RIPA buffer with protease inhibitors; standardize tissue:buffer ratios; maintain samples at 4°C during processing.

• Band Size Variations:

  • Challenge: DNAJB9 may appear between 26-30 kDa depending on post-translational modifications.

  • Solution: Include positive control samples; use protein ladders with clear markers in this range .

• Antibody Specificity:

  • Challenge: Cross-reactivity with other DNAJ family proteins.

  • Solution: Use validated antibodies; consider pre-absorption controls; compare results with multiple antibodies targeting different epitopes.

• Transfer Efficiency Problems:

  • Challenge: Poor transfer of DNAJB9 to membranes.

  • Solution: Optimize transfer conditions (time, voltage, buffer composition); use PVDF membranes for better protein retention.

• Protocol Optimization:

  • Challenge: Finding optimal dilution and incubation conditions.

  • Solution: Follow manufacturer protocols but titrate antibody concentration; most DNAJB9 antibodies work optimally at 1:500-1:1000 dilution for WB .

• Signal Development:

  • Challenge: Weak or excessive signal.

  • Solution: For weak signals, consider longer exposure times or more sensitive detection systems; for excessive signals, further dilute primary antibody or reduce exposure time.

Documentation of all variables across experiments will facilitate troubleshooting and protocol refinement.

What considerations are important when using DNAJB9 antibodies for co-localization studies with other proteins?

Co-localization studies with DNAJB9 and other proteins require careful methodological considerations:

• Antibody Compatibility:

  • Challenge: Primary antibodies must be from different host species for simultaneous detection.

  • Solution: Plan combinations carefully; for example, use rabbit anti-DNAJB9 with mouse anti-IgG for co-localization studies in FGN .

• Fluorophore Selection:

  • Challenge: Spectral overlap can give false co-localization signals.

  • Solution: Choose widely separated fluorophores; consider linear unmixing for closely spaced emissions; include single-label controls.

• Sequential vs. Simultaneous Staining:

  • Challenge: Some epitopes may be masked in simultaneous protocols.

  • Solution: Compare sequential and simultaneous staining approaches; for DNAJB9 and IgG co-localization in FGN, successful dual IF staining has been documented .

• Cross-Reactivity Control:

  • Challenge: Secondary antibodies may cross-react with inappropriate primaries.

  • Solution: Include secondary-only controls; perform single-primary controls with both secondaries to check cross-reactivity.

• Quantitative Analysis:

  • Challenge: Visual assessment of co-localization is subjective.

  • Solution: Use digital image analysis with standard co-localization coefficients (Manders, Pearson); set consistent thresholds across samples.

• Tissue Preparation Consistency:

  • Challenge: Different fixation or antigen retrieval can affect different epitopes unequally.

  • Solution: Standardize all preprocessing steps; validate protocols on known positive controls.

Following these considerations will improve the reliability of co-localization data, particularly in complex disease contexts like FGN.

What are emerging applications of DNAJB9 antibodies beyond FGN diagnosis?

DNAJB9 antibody applications are expanding beyond FGN diagnosis into several promising areas:

• Transplant Monitoring: Emerging evidence suggests DNAJB9 antibodies may be valuable for monitoring FGN recurrence in kidney transplants, potentially informing immunosuppression strategies .

• Cancer Research: With DNAJB9's roles in ER stress responses and protein quality control, antibodies are being applied to study its expression in various cancers, including breast and prostate cancer tissues .

• Cell Stress Biology: DNAJB9 antibodies are increasingly used to monitor cellular stress responses, particularly in models of ER stress and the unfolded protein response pathway.

• Neurodegenerative Disease Research: Given the role of protein misfolding in neurodegenerative conditions, researchers are exploring DNAJB9 expression patterns in these contexts.

• Therapeutic Development: As understanding of DNAJB9's role in FGN pathogenesis advances, antibodies may help evaluate potential therapeutic targets in the pathway.

• Non-Invasive Diagnostics Development: Research into serum DNAJB9 levels may lead to less invasive diagnostic approaches for FGN, utilizing antibodies in immunoassay formats.

These emerging applications highlight the expanding importance of DNAJB9 antibodies beyond their established diagnostic role.

How might advances in antibody engineering impact future DNAJB9 detection methods?

Advances in antibody engineering are likely to transform DNAJB9 detection in several ways:

• Single-Domain Antibodies: Development of nanobodies or single-domain antibodies against DNAJB9 may improve tissue penetration and provide higher resolution in immunohistochemistry and immunoelectron microscopy.

• Multispecific Antibodies: Bispecific or multispecific antibodies that simultaneously target DNAJB9 and other FGN-associated proteins could provide more comprehensive disease profiling in a single test.

• Site-Specific Conjugation: Precisely engineered antibody-dye conjugates with defined dye-to-antibody ratios will improve quantitative analysis of DNAJB9 expression levels.

• Recombinant Antibody Fragments: Fab and scFv fragments with enhanced tissue penetration may improve detection in challenging samples or thick tissue sections.

• Automated Detection Systems: Integration of DNAJB9 antibodies into digital pathology platforms with AI-assisted image analysis could standardize interpretation and reduce inter-observer variability.

• Point-of-Care Applications: Development of rapid immunochromatographic tests using engineered DNAJB9 antibody fragments could expand testing accessibility.

These technological advances will likely improve sensitivity, specificity, and clinical utility of DNAJB9 detection methods.

What unresolved questions remain regarding the pathogenic role of DNAJB9 in fibrillary glomerulonephritis?

Despite significant advances, several crucial questions about DNAJB9's role in FGN pathogenesis remain unanswered:

• Origin of DNAJB9 in Deposits: It remains unclear whether the DNAJB9 in glomerular deposits originates from local production, circulating sources, or both. Research has shown no significant upregulation of DNAJB9 mRNA in glomeruli despite protein accumulation .

• Fibrillogenesis Mechanism: The process by which DNAJB9 contributes to fibril formation remains unresolved. Does DNAJB9 directly nucleate fibrils, or does it bind to pre-formed fibrils initiated by other factors?

• Relationship with Immunoglobulins: While DNAJB9 co-localizes with IgG in deposits, the nature of this interaction is not fully understood. Is it a direct binding interaction, and what mediates it?

• Conformational Changes: Whether DNAJB9 undergoes conformational changes during FGN development, potentially exposing cryptic epitopes or aggregation-prone regions, remains to be determined.

• Genetic Contributions: Although initial studies have not found pathogenic mutations, more comprehensive genomic analyses may reveal subtle genetic contributions to DNAJB9 dysfunction in FGN .

• Therapeutic Targeting: Whether DNAJB9 itself represents a viable therapeutic target, or if intervention should focus on upstream or downstream pathways, remains an open question requiring further research.