DNAJB1 Antibody

What is the primary cellular function of DNAJB1 and why is it a target for antibody-based research?

DNAJB1 functions as a molecular chaperone that stimulates the ATPase activity of Hsp70 heat-shock proteins to promote proper protein folding and prevent misfolded protein aggregation . It contains a highly conserved amino acid sequence called the 'J-domain' that characterizes the DnaJ/Hsp40 family . DNAJB1 is involved in wide-ranging cellular events including protein folding, oligomeric protein complex assembly, and stress response mechanisms .

The protein negatively regulates heat shock-induced HSF1 transcriptional activity during the attenuation and recovery phase of heat shock response . Its role in protein quality control makes it a valuable research target for understanding fundamental cellular processes and disease mechanisms, particularly in neurodegenerative disorders and cancer where protein misfolding is implicated.

What are the recommended applications for DNAJB1 antibodies in cellular research?

Based on validated research applications, DNAJB1 antibodies are particularly effective for:

• Immunofluorescence at 0.25-2 μg/mL concentration to visualize subcellular localization and expression patterns

• Immunohistochemistry at 1:200-1:500 dilution for tissue-specific expression analysis

• Western blotting to detect DNAJB1 protein expression levels in cell lines and tissue samples

• Co-immunoprecipitation experiments to study protein-protein interactions, particularly with Hsp70 family proteins

When designing experiments, researchers should validate antibody specificity using appropriate positive controls (such as CCA cell lines which show high DNAJB1 expression) and negative controls (such as HiBECs cell lines which show low expression) .

How can researchers validate the specificity of DNAJB1 antibodies?

A multi-tiered validation approach is recommended:

• Orthogonal validation: Compare antibody results with RNAseq data to confirm correlation between protein and mRNA levels

• Positive/negative tissue controls: Test antibodies on tissues known to express DNAJB1 at high levels (e.g., CCA cell lines) versus those with minimal expression (e.g., HiBECs)

• siRNA knockdown: Reduce DNAJB1 expression and confirm corresponding decrease in antibody signal

• Western blot analysis: Confirm single band at expected molecular weight (approximately 40 kDa)

• Immunofluorescence localization: Verify predominant cytoplasmic localization pattern consistent with DNAJB1's known distribution

For comprehensive validation, compare results across multiple detection methods. In ICC studies, DNAJB1 should show strong cytoplasmic staining in positive cell lines and weak or no staining in negative controls .

What are the optimal conditions for using DNAJB1 antibodies in immunohistochemistry?

For optimal immunohistochemistry results with DNAJB1 antibodies:

For formalin-fixed, paraffin-embedded (FFPE) samples, ensure complete deparaffinization and sufficient antigen retrieval to expose the epitope. When analyzing results, note that DNAJB1 shows predominantly cytoplasmic localization , so nuclear staining may indicate non-specific binding or cross-reactivity.

What are the key considerations for using DNAJB1 antibodies in various buffer systems?

Buffer composition significantly impacts DNAJB1 antibody performance across different applications:

• Storage buffer: Commercial DNAJB1 antibodies are typically supplied in buffered aqueous glycerol solutions . Avoid repeated freeze-thaw cycles which can degrade antibody quality.

• Western blot buffers:

  • Lysis buffer: RIPA buffer supplemented with protease inhibitors is effective

  • Transfer buffer: Standard Tris-glycine with 20% methanol works well

  • Blocking buffer: 5% non-fat dry milk in TBST shows good results with minimal background

• Immunofluorescence buffers:

  • Fixation: 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilization: 0.1-0.5% Triton X-100 in PBS for 5-10 minutes

  • Blocking: 1-5% BSA or normal serum in PBS with 0.1% Tween-20

• Immunoprecipitation buffers:

  • Lysis buffer: NP-40 or CHAPS-based buffers maintain protein-protein interactions

  • Wash buffers: Moderate stringency to reduce non-specific binding while preserving specific interactions

The pH range of 7.2-7.4 is optimal for most applications. For cross-linking experiments investigating DNAJB1 interactions (like those with HTTExon1Q48), specialized cross-linking buffers may be required .

How can researchers minimize background and non-specific binding when using DNAJB1 antibodies?

To reduce background and enhance signal-to-noise ratio:

• Optimize blocking conditions: Use 5% normal serum or BSA from a species different from the primary antibody host (rabbit in this case)

• Antibody titration: Perform dilution series to determine optimal concentration that maximizes specific signal while minimizing background

• Washing protocol optimization:

  • Increase number of washes (minimum 3-5 washes)

  • Extend washing time (5-10 minutes per wash)

  • Include 0.1-0.3% Tween-20 or Triton X-100 in wash buffers

• Pre-absorption controls: Pre-incubate primary antibody with immunizing peptide to confirm specificity

• Secondary antibody selection: Use highly cross-adsorbed secondary antibodies specific to rabbit IgG to minimize cross-reactivity

• Tissue autofluorescence reduction: For immunofluorescence applications, treat sections with sodium borohydride or commercial autofluorescence quenching reagents

• Endogenous peroxidase quenching: For IHC-DAB applications, block endogenous peroxidases with 0.3% H₂O₂ in methanol

The immunogen sequence used for DNAJB1 antibody generation (DPHAMFAEFFGGRNPFDTFFGQRNGEEGMDIDDPFSGFPMGMGGFTNVNFGRSRSAQEPARKKQDPPVTHDL) can help researchers identify potential cross-reactivity with related proteins.

How can DNAJB1 antibodies be utilized to study its role in cancer pathogenesis, particularly in cholangiocarcinoma?

DNAJB1 has emerged as a significant biomarker in cholangiocarcinoma (CCA) research. Methodological approaches include:

• Expression profiling: DNAJB1 antibodies can quantify upregulation in CCA tissues compared to normal biliary epithelium. RT-qPCR, western blot, and immunohistochemistry have shown that DNAJB1 is significantly overexpressed in CCA cell lines compared to normal HiBECs .

• Clinicopathological correlation: Researchers have demonstrated associations between DNAJB1 expression and several clinicopathological parameters:

  • Pathological differentiation

  • Vessel invasion

  • T stage

  • Lymph node metastasis

  • Clinical stage

• Prognostic marker analysis: Kaplan-Meier survival analysis with log-rank testing has shown that DNAJB1 upregulation correlates with poor prognosis in CCA patients .

• Bile biomarker validation: DNAJB1 can be detected in bile samples, making it potentially valuable as a non-invasive diagnostic marker .

• Mechanistic studies: Co-immunoprecipitation with DNAJB1 antibodies can identify interaction partners involved in cancer-related pathways, particularly those involving protein quality control mechanisms.

For CCA research, it's recommended to compare DNAJB1 expression across multiple CCA cell lines (e.g., QBC939, RBE, HCCC-9810, and CCLP-1) against normal biliary epithelial controls .

What techniques can be employed to study the interaction between DNAJB1 and Huntingtin protein using antibodies?

The interaction between DNAJB1 and Huntingtin (HTT) protein, particularly mutant forms with expanded polyglutamine repeats, represents an important research area. Advanced methodological approaches include:

• Cross-linking mass spectrometry (XL-MS): This technique has successfully identified specific interactions between DNAJB1/Hsc70 and HTTExon1Q48 . Researchers should use membrane-permeable crosslinkers followed by immunoprecipitation with DNAJB1 antibodies and mass spectrometry analysis.

• Site-directed mutagenesis studies: Mutation of the HTT-binding motif (HBM) in DNAJB1, particularly the highly conserved H244 residue, completely abrogates suppression and disaggregation of HTT fibrils . Researchers can generate DNAJB1 H244A mutants to study binding specificity.

• Co-immunoprecipitation assays: DNAJB1 antibodies can pull down HTT protein complexes, allowing researchers to study interactions with both soluble and aggregated forms.

• Cellular aggregation models: In HEK293 cells overexpressing HTTExon1Q97, wild-type DNAJB1 suppresses aggregation while DNAJB1 H244A does not . Researchers can use immunofluorescence with DNAJB1 antibodies to track co-localization with HTT aggregates.

• Molecular dynamics simulation validation: Computational predictions of DNAJB1-HTT interactions can be verified experimentally using antibody-based techniques .

The key insight from these studies is that DNAJB1 binds to the polyproline-rich domain (PRD) of HTT through a specific binding motif, providing a shared binding site in both soluble and aggregated forms .

How can researchers optimize DNAJB1 antibody-based assays for studying heat shock response mechanisms?

DNAJB1 plays a crucial role in heat shock response as a co-chaperone that regulates HSF1 activity . To study these mechanisms:

• Time-course experiments: Design protocols with precise temperature control and timing:

  • Expose cells to heat shock (42-45°C for 30-60 minutes)

  • Collect samples at multiple time points during recovery (0, 1, 2, 4, 8, 24 hours)

  • Use DNAJB1 antibodies to track expression changes via western blot or immunofluorescence

• Co-localization studies: Use dual immunofluorescence with antibodies against:

  • DNAJB1

  • HSP70 family proteins

  • HSF1 transcription factor

  This reveals dynamic spatial relationships during stress response phases.

• Chromatin immunoprecipitation (ChIP) assays: While not directly using DNAJB1 antibodies, these can be coupled with DNAJB1 knockdown/overexpression to study its impact on HSF1 binding to heat shock elements.

• Functional assays: Measure DNAJB1's effect on:

  • HSP70 ATPase activity using purified proteins

  • Protein aggregation using fluorescence-based assays

  • Cell survival following heat stress

• Quantitative image analysis: For immunofluorescence experiments, implement rigorous quantification:

  • Measure nuclear vs. cytoplasmic DNAJB1 distribution

  • Quantify co-localization coefficients with HSP70

  • Track changes in aggregate formation/resolution

When analyzing results, remember that DNAJB1 negatively regulates heat shock-induced HSF1 transcriptional activity during the attenuation and recovery phase , so expression patterns and localizations will change dynamically throughout the heat shock response cycle.

What are the common challenges encountered when using DNAJB1 antibodies and how can they be addressed?

Researchers frequently encounter these challenges when working with DNAJB1 antibodies:

• Cross-reactivity with related proteins:

  • Challenge: DNAJB1 belongs to the large DnaJ/Hsp40 family with highly conserved domains.

  • Solution: Use antibodies targeting unique regions outside the conserved J-domain. The antibody raised against the immunogen sequence DPHAMFAEFFGGRNPFDTFFGQRNGEEGMDIDDPFSGFPMGMGGFTNVNFGRSRSAQEPARKKQDPPVTHDL targets a more specific region .

• Variable expression levels across tissues:

  • Challenge: DNAJB1 expression varies significantly between tissue types.

  • Solution: Include appropriate positive controls (e.g., heat-shocked cells) and negative controls (knockdown samples) for each experiment type.

• Detection in fixed tissues:

  • Challenge: Epitope masking during fixation.

  • Solution: Optimize antigen retrieval methods; citrate buffer (pH 6.0) heat-induced retrieval works well for most DNAJB1 epitopes.

• High background in immunofluorescence:

  • Challenge: Non-specific binding in cytoplasm.

  • Solution: Increase blocking time (2 hours minimum), use higher BSA concentrations (3-5%), and add 0.1-0.3% Triton X-100 to wash buffers.

• Inconsistent western blot results:

  • Challenge: Multiple bands or weak signal.

  • Solution: Use fresh lysates, optimize transfer conditions for mid-sized proteins (30-50 kDa), and consider enhanced chemiluminescence detection for improved sensitivity.

• Co-detection with interaction partners:

  • Challenge: Antibody interference in co-IP experiments.

  • Solution: Use antibody crosslinking to protein A/G beads to prevent heavy chain contamination in immunoprecipitation eluates.

When troubleshooting, always refer to the recommended concentrations (0.25-2 μg/mL for IF, 1:200-1:500 for IHC) as starting points and adjust based on specific experimental conditions.

How can researchers confirm that their DNAJB1 antibody is recognizing the intended target in experimental systems?

A comprehensive validation strategy should include:

• Western blot molecular weight verification:

  • Expected size: Human DNAJB1 migrates at approximately 38-40 kDa

  • Compare with recombinant DNAJB1 protein as size control

  • Validate in multiple cell types with known expression levels

• Genetic validation approaches:

  • siRNA/shRNA knockdown: Confirm signal reduction proportional to knockdown efficiency

  • CRISPR/Cas9 knockout: Complete signal loss in null cells

  • Overexpression: Increased signal intensity in transfected cells

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Observe elimination of specific signal while non-specific binding remains

• Orthogonal method comparison:

  • Compare antibody results with RNAseq or proteomics data

  • Verify correlation between protein and mRNA expression levels across samples

• Cross-species reactivity testing:

  • If antibody is claimed to work across species, verify in each species

  • Compare expression patterns with known species-specific differences

• Domain-specific verification:

  • For functional studies of DNAJB1 domains (e.g., the HTT-binding motif), generate domain mutants

  • Confirm that antibodies to different domains show concordant results

• Mass spectrometry validation:

  • Immunoprecipitate DNAJB1 and perform MS analysis

  • Confirm peptide sequence matches expected target

This multi-method validation approach ensures research reliability and reproducibility when working with DNAJB1 antibodies.

What controls are essential when using DNAJB1 antibodies in various experimental applications?

Implementing appropriate controls is critical for experimental rigor:

For Western Blotting:

• Positive control: Cell lysate with confirmed DNAJB1 expression (e.g., CCA cell lines)

• Negative control: DNAJB1 knockdown/knockout cell lysate

• Loading control: Housekeeping protein (β-actin, GAPDH)

• Molecular weight marker: To confirm correct band size (~38-40 kDa)

• Secondary antibody-only control: To detect non-specific binding

For Immunohistochemistry/Immunofluorescence:

• Positive tissue control: Tissues known to express DNAJB1 (liver, brain)

• Negative tissue control: Tissues with minimal expression or DNAJB1-negative regions

• Isotype control: Non-specific IgG from same species as primary antibody

• Blocking peptide control: Primary antibody pre-incubated with immunizing peptide

• Secondary antibody-only control: To assess background

For Co-immunoprecipitation:

• Input control: Sample of lysate before immunoprecipitation

• IgG control: Non-specific IgG from same species as DNAJB1 antibody

• Reverse co-IP: Immunoprecipitate with antibody against interacting protein

• Positive interaction control: Known DNAJB1 binding partner (e.g., HSP70)

• Negative interaction control: Protein not expected to interact with DNAJB1

For Functional Assays:

• Heat shock induction control: Samples with verified DNAJB1 upregulation

• Pharmacological controls: Compounds that modulate chaperone activity

• Time-course controls: Multiple time points to capture dynamic changes

• Wild-type vs. mutant comparisons: Especially for studies of the HTT-binding motif (H244A mutant)

These controls ensure experimental validity and facilitate troubleshooting if unexpected results occur.

How can DNAJB1 antibodies contribute to research on fibrolamellar carcinoma and the DNAJB1-PRKACA fusion protein?

Fibrolamellar carcinoma (FLC) is characterized by a recurrent genomic deletion resulting in the DNAJB1-PRKACA chimeric transcript, presenting unique research opportunities:

• Detection strategies for the fusion protein:

  • Western blotting: Use DNAJB1 antibodies targeting the N-terminal region retained in the fusion protein

  • Immunohistochemistry: Compare staining patterns between DNAJB1 and PRKACA antibodies to identify cells expressing the fusion

  • Dual-color FISH: Combine with genomic approaches to confirm fusion at the DNA level

• Differential diagnosis applications:

  • The DNAJB1-PRKACA chimeric transcript is elevated in 100% of FLC cases, making it a valuable diagnostic marker

  • Develop immunohistochemistry protocols that distinguish wild-type DNAJB1 from the fusion protein

  • Compare expression in FLC versus conventional hepatocellular carcinoma

• Functional studies of pathogenesis:

  • Use co-immunoprecipitation with DNAJB1 antibodies to identify altered interaction partners of the fusion protein

  • Employ proximity ligation assays to visualize protein-protein interactions in situ

  • Develop cell models expressing the fusion to study downstream effects on chaperone networks

• Therapeutic target exploration:

  • Screen for compounds that specifically interfere with fusion protein function

  • Test antibody-drug conjugates targeting the unique epitopes created at the fusion junction

  • Explore the disruption of critical interactions specific to the fusion protein

This research area highlights how DNAJB1 antibodies can be repurposed from studying wild-type protein function to investigating disease-specific alterations with diagnostic and therapeutic implications.

What methodologies can researchers employ to study DNAJB1's role in neurodegenerative diseases using antibodies?

DNAJB1's role in protein quality control makes it particularly relevant to neurodegenerative disease research:

• Huntington's disease models:

  • The DNAJB1-Hsc70-Apg2 chaperone complex can both suppress aggregation and disaggregate preformed HTT amyloid fibrils

  • Use immunofluorescence with DNAJB1 antibodies to track co-localization with HTT aggregates in cellular models

  • Implement proximity ligation assays to confirm direct interaction with the polyproline-rich domain (PRD) of HTT

  • Study the effect of DNAJB1 H244A mutation, which completely abrogates suppression and disaggregation of HTT fibrils

• Alzheimer's disease applications:

  • Examine DNAJB1 interaction with tau and amyloid-β using co-immunoprecipitation

  • Quantify DNAJB1 levels in different brain regions from AD patient samples

  • Investigate whether DNAJB1 overexpression affects tau or amyloid pathology

• Parkinson's disease studies:

  • Explore DNAJB1 interaction with α-synuclein using biochemical and imaging approaches

  • Examine DNAJB1 levels in dopaminergic neurons in disease models

  • Test whether DNAJB1 modulation affects α-synuclein aggregation kinetics

• ALS/FTD research:

  • Investigate DNAJB1 interactions with TDP-43 and FUS

  • Determine if DNAJB1 expression correlates with disease progression

  • Test modulation of DNAJB1 activity as a therapeutic approach

• Methodological considerations for neuronal systems:

  • Optimize fixation protocols for neuronal cultures to preserve DNAJB1 epitope accessibility

  • Implement tissue clearing techniques for improved visualization in brain sections

  • Develop fractionation protocols to separate soluble and aggregated protein species

The HTT-binding motif (HBM) in DNAJB1 represents a promising target for therapeutic development focused on HD and potentially other polyglutamine diseases .

How can researchers utilize DNAJB1 antibodies to investigate its role in cellular stress response pathways beyond heat shock?

DNAJB1 functions extend beyond classical heat shock to multiple stress response mechanisms:

• Endoplasmic reticulum (ER) stress research:

  • DNAJB1 has been implicated in ER stress response mechanisms

  • Use immunofluorescence to track DNAJB1 redistribution during ER stress induction with tunicamycin or thapsigargin

  • Combine with markers of ER stress (BiP/GRP78, XBP1 splicing) to correlate with DNAJB1 activity

  • Quantify changes in DNAJB1 phosphorylation state during unfolded protein response activation

• Oxidative stress pathway analysis:

  • Compare DNAJB1 expression and localization after treatment with oxidative stress inducers (H₂O₂, paraquat)

  • Co-immunoprecipitate to identify oxidative stress-specific interaction partners

  • Assess whether DNAJB1 knockdown sensitizes cells to oxidative damage

• Proteotoxic stress models:

  • Study DNAJB1 involvement in the proteasome pathway

  • Use proteasome inhibitors (MG132, bortezomib) and track DNAJB1 recruitment to aggresomes

  • Implement live-cell imaging with fluorescently tagged DNAJB1 to monitor dynamics during stress

• Autophagy connections:

  • DNAJB1 serves as an autophagy-associated protein involved in tumor development

  • Investigate co-localization with autophagy markers (LC3, p62) during stress conditions

  • Determine if DNAJB1 influences selective autophagy of protein aggregates

• Viral infection responses:

  • DNAJB1 has been linked to viral infection processes

  • Examine changes in DNAJB1 expression and localization during viral infection

  • Investigate potential direct interactions with viral proteins

• Integration of multi-stress responses:

  • Design experiments comparing DNAJB1 dynamics across different stressors

  • Develop quantitative assays to measure DNAJB1 chaperone activity under varied stress conditions

  • Create stress response "signatures" based on DNAJB1 interactions and modifications

These approaches allow researchers to build a comprehensive understanding of DNAJB1's role in integrated cellular stress response networks beyond the classical heat shock paradigm.

What are the most effective approaches for studying post-translational modifications of DNAJB1 using antibody-based techniques?

Post-translational modifications (PTMs) regulate DNAJB1 function in complex ways:

• Phosphorylation analysis:

  • Use phospho-specific antibodies targeting known or predicted DNAJB1 phosphorylation sites

  • Implement Phos-tag SDS-PAGE followed by western blotting with DNAJB1 antibodies to resolve phosphorylated species

  • Compare phosphorylation patterns before and after stress induction

  • Couple with kinase inhibitors to identify regulatory pathways

• Ubiquitination detection:

  • Perform immunoprecipitation with DNAJB1 antibodies followed by ubiquitin western blotting

  • Use tandem ubiquitin binding entities (TUBEs) pulldown followed by DNAJB1 detection

  • Implement proximity ligation assays between DNAJB1 and ubiquitin

  • Study proteasomal degradation kinetics with cycloheximide chase experiments

• Acetylation studies:

  • Use pan-acetyl-lysine antibodies following DNAJB1 immunoprecipitation

  • Compare acetylation patterns with and without HDAC inhibitors

  • Implement mass spectrometry to identify specific acetylation sites

• SUMOylation analysis:

  • Perform SUMO-IP followed by DNAJB1 western blotting

  • Use SUMO-site prediction tools to generate mutants for functional testing

  • Employ in vitro SUMOylation assays with recombinant proteins

• Methods for studying PTM crosstalk:

  • Sequential immunoprecipitation with different PTM-specific antibodies

  • Mass spectrometry analysis of purified DNAJB1 to identify co-occurring modifications

  • Generate site-specific mutants to assess functional impact of PTM combinations

• Spatiotemporal PTM dynamics:

  • Implement FRET-based sensors to monitor real-time changes in DNAJB1 conformation

  • Use super-resolution microscopy to track differently modified DNAJB1 subpopulations

  • Develop pulse-chase labeling strategies to follow modification/demodification cycles

When designing experiments, remember that PTMs likely influence DNAJB1's ability to interact with client proteins and co-chaperones, potentially explaining context-specific functions in different cellular environments.

How can advanced imaging techniques enhance DNAJB1 antibody-based visualization in complex cellular contexts?

Cutting-edge imaging approaches provide unprecedented insights into DNAJB1 biology:

• Super-resolution microscopy applications:

  • Structured Illumination Microscopy (SIM): Visualize DNAJB1 distribution relative to cellular organelles

  • Stochastic Optical Reconstruction Microscopy (STORM): Achieve nanoscale resolution of DNAJB1 clustering during stress

  • Stimulated Emission Depletion (STED): Detect fine structural details of DNAJB1-containing complexes

  Optimization tip: Use primary DNAJB1 antibodies conjugated to bright, photostable fluorophores or high-quality secondary antibodies with minimal size.

• Live-cell imaging strategies:

  • Antibody fragment (Fab) labeling: Monitor DNAJB1 dynamics in living cells

  • SNAP/CLIP tag fusions: Orthogonal labeling of DNAJB1 with synthetic dyes

  • Fluorescence Recovery After Photobleaching (FRAP): Measure DNAJB1 mobility in different cellular compartments

• Multiplexed imaging approaches:

  • Cyclic Immunofluorescence (CycIF): Analyze DNAJB1 alongside dozens of other proteins

  • Mass Cytometry Imaging (IMC): Simultaneous visualization of over 40 proteins including DNAJB1

  • DNA-Paint: Achieve multiplexed super-resolution using DNA-conjugated antibodies

• Correlative light-electron microscopy (CLEM):

  • Immunogold labeling of DNAJB1 for transmission electron microscopy

  • Preserve fluorescence signal through specialized fixation for correlative analysis

  • Track DNAJB1 association with specific ultrastructural features

• Expansion microscopy opportunities:

  • Physical expansion of specimens allows super-resolution on conventional microscopes

  • Optimize DNAJB1 antibody concentration for expanded samples (typically lower concentrations)

  • Implement iterative expansion for ultra-high resolution

• Tissue-specific visualization enhancements:

  • Tissue clearing techniques (CLARITY, iDISCO) for deep tissue imaging

  • Serial section reconstruction for 3D visualization of DNAJB1 distribution

  • Spatial transcriptomics correlation with protein distribution

DNAJB1's predominantly cytoplasmic localization can be more precisely characterized using these advanced techniques, potentially revealing microdomains of activity or association with specific organelles during different cellular states.