DNAJA3 Antibody

What is DNAJA3 and what cellular functions does it regulate?

DNAJA3, also known as tumorous imaginal disc (Tid1), hTID-1, HCA57, or DnaJ homolog subfamily A member 3, functions as a tumor suppressor implicated in lymphocyte development and survival . This 52.5 kilodalton protein plays crucial roles in T and B cell development, with significant effects on mitochondrial function . Research has demonstrated that DNAJA3 also possesses antiviral properties, particularly against foot-and-mouth disease virus (FMDV) by interacting with viral VP1 protein and triggering its degradation via the lysosomal pathway .

What applications are DNAJA3 antibodies validated for in research?

DNAJA3 antibodies have been validated for multiple research applications with varying sensitivities:

Different antibody clones show varying performance across these applications . When selecting an antibody, researchers should review validation data showing specificity in their particular application and target species.

How should researchers plan experiments using DNAJA3 antibodies with appropriate controls?

Experimental design for DNAJA3 antibody-based research requires careful consideration of controls:

• Positive controls: Include tissues/cells with known DNAJA3 expression (lymphoid tissues, mitochondria-rich cells)

• Negative controls: DNAJA3 knockout samples or siRNA-treated cells

• Antibody controls: Include isotype controls and secondary antibody-only controls

• Cross-validation: Use multiple antibody clones targeting different epitopes when possible

• Species verification: Confirm reactivity with your species of interest (human, mouse, rat, etc.)

For Western blot applications, verify signal at the expected molecular weight (52.5 kDa), and consider blocking peptide competition assays to confirm specificity.

How can researchers effectively investigate DNAJA3's role in B cell development?

B cell development studies require specialized methodological approaches:

• Model selection: Consider B cell-specific DNAJA3 knockout models (e.g., CD19-Cre/+; DNAJA3 flx/flx) for in vivo studies

• Cell population analysis: Utilize flow cytometry with B cell developmental markers (B220, CD19, IgM, IgD) alongside DNAJA3 antibodies

• Functional assays: Measure B cell blastogenesis (CFSE dilution, MTT assays) and immunoglobulin production (ELISA) to assess functional impact

• Mitochondrial analysis: Evaluate mitochondrial mass, membrane potential, and respiratory complex proteins, as DNAJA3 deficiency impacts these parameters

What methodological approaches enable studying DNAJA3's role in mitochondrial function?

Investigating DNAJA3's mitochondrial functions requires specialized techniques:

• Subcellular fractionation: Use differential centrifugation to isolate mitochondrial fractions

• Respiratory analysis: Employ Seahorse assays to measure oxygen consumption rate

• Membrane potential: Utilize JC-1 or TMRM dyes to assess mitochondrial membrane potential

• Complex protein quantification: Western blot analysis of OXPHOS protein complexes

• Microscopy: Confocal imaging with mitochondrial markers (TOM20, COX IV) for co-localization

Studies have shown DNAJA3 deficiency significantly increases dysfunctional mitochondrial activity while decreasing mitochondrial mass, membrane potential, and mitochondrial respiratory complex proteins .

How does DNAJA3 contribute to antiviral responses, and how can researchers investigate this function?

DNAJA3 exhibits significant antiviral activity through multiple mechanisms:

• Viral protein interaction: DNAJA3 directly interacts with viral proteins such as FMDV VP1

• Lysosomal degradation: DNAJA3 can trigger degradation of viral proteins via the lysosomal pathway

• IFN-β signaling regulation: DNAJA3 abrogates viral protein-induced inhibition of IFN-β signaling

Research methodologies to investigate these functions include:

• Co-immunoprecipitation to detect protein-protein interactions

• Confocal microscopy for co-localization studies

• Lysosomal inhibitor treatments to confirm degradation pathways

• IRF3 phosphorylation, dimerization, and nuclear translocation assays to assess interferon signaling

Overexpression of DNAJA3 dramatically dampens FMDV replication, whereas loss of function of DNAJA3 elicits opposing effects .

How can researchers address weak or inconsistent DNAJA3 signal detection?

Signal optimization strategies include:

Optimization should be systematic, changing one variable at a time while documenting outcomes.

How should researchers interpret discrepancies between different DNAJA3 antibody clones?

When facing discrepant results:

• Review epitope information: Different domains may show different expression patterns

• Consider isoforms: DNAJA3 has multiple isoforms that may be differentially recognized

• Evaluate post-translational modifications: These may affect epitope accessibility

• Validate with orthogonal methods: Confirm with mRNA quantification or genetic approaches

• Document experimental conditions: Identify variables that might explain discrepancies

Cross-validation with multiple antibodies targeting different epitopes can provide more comprehensive insights into DNAJA3 expression and localization.

What special considerations apply when studying DNAJA3 in different immune cell populations?

Cell-specific considerations include:

• Isolation protocols: Use appropriate methods for isolating B cells (CD19+ selection) or T cells (CD3+ selection)

• Purity verification: Confirm population purity (~98%) by flow cytometry before analysis

• Developmental stages: Consider stage-specific markers for developmental studies

• Activation status: Account for changes in DNAJA3 expression during immune cell activation

• Tissue source: Different lymphoid tissues may show variable DNAJA3 expression patterns

For DNAJA3 knockout studies, verification of deletion should be performed at both protein (immunoblotting) and genomic (PCR) levels .

How can DNAJA3 antibodies be utilized in cancer research applications?

DNAJA3 functions as a tumor suppressor, offering several research avenues:

• Expression correlation: Compare DNAJA3 levels across normal and malignant tissues

• Prognostic potential: Correlate expression with patient outcomes in tissue microarrays

• Mechanism studies: Investigate interactions with known oncogenes and tumor suppressors

• Therapeutic targeting: Explore modulation of DNAJA3 pathways as intervention strategies

• Biomarker development: Assess DNAJA3 as a diagnostic or predictive biomarker

Researchers should employ multiple detection methods (IHC, WB, IF) for comprehensive characterization.

What considerations are important when designing multi-parameter flow cytometry panels including DNAJA3?

Panel design recommendations:

• Fluorophore selection: Choose fluorophores with minimal spectral overlap

• Compensation controls: Include single-stained controls for each fluorochrome

• Antibody titration: Determine optimal concentration for each antibody

• Fixation compatibility: Validate DNAJA3 antibody performance with your fixation protocol

• Intracellular staining: Optimize permeabilization conditions for intracellular detection

• Co-expression analysis: Include relevant lineage and functional markers

For B cell studies, consider combining DNAJA3 with markers like B220, CD19, IgM, and IgD to identify developmental stages .

How should researchers approach studying DNAJA3 interactions with other cellular proteins?

Protein interaction methodologies include:

• Co-immunoprecipitation: Pull down DNAJA3 and identify interacting partners

• Proximity ligation assay: Visualize protein interactions in situ

• FRET/BRET analysis: Measure real-time interactions in living cells

• Mass spectrometry: Identify interaction partners in an unbiased manner

• Domain mapping: Create truncation mutants to identify interaction domains

• Functional validation: Confirm biological relevance of interactions

These approaches can identify novel binding partners beyond the known viral interactions like FMDV VP1 .

What emerging technologies might enhance DNAJA3 research?

Future directions in DNAJA3 antibody research may include:

• Single-cell approaches: Analyze DNAJA3 expression at single-cell resolution

• Spatial transcriptomics: Map DNAJA3 expression within tissue microenvironments

• CRISPR screens: Identify genes that modify DNAJA3 function

• Nanobody development: Create smaller antibody formats for improved imaging

• Therapeutic antibodies: Develop antibodies that modulate DNAJA3 function

These technologies will provide deeper insights into DNAJA3's diverse cellular functions.

How can researchers integrate multi-omics approaches in DNAJA3 studies?

Integrative strategies include:

• Transcriptomics + proteomics: Correlate mRNA and protein expression patterns

• Proteomics + interactomics: Identify and validate protein interaction networks

• Metabolomics + functional assays: Connect metabolic changes to DNAJA3 function

• Epigenomics + expression analysis: Identify regulatory mechanisms controlling DNAJA3

• Systems biology modeling: Integrate multiple data types to predict DNAJA3 function

This multi-dimensional approach can provide comprehensive understanding of DNAJA3's role in cellular processes.