ERDJ3B Antibody

What is ERdj3 and why is it significant in cellular research?

ERdj3 (also known as DNAJB11) is a soluble glycoprotein located in the endoplasmic reticulum (ER) lumen that functions as a co-chaperone of binding immunoglobulin protein (BiP), a 70 kilodalton heat shock protein chaperone required for proper protein folding and assembly in the ER. The protein contains a highly conserved J domain of approximately 70 amino acids with a characteristic His-Pro-Asp (HPD) motif, which is critical for its function. ERdj3 may regulate BiP activity by stimulating its ATPase activity, thereby playing an essential role in protein quality control mechanisms within the ER . Its significance in cellular research stems from its involvement in the unfolded protein response (UPR), ER stress, and protein folding pathways that are implicated in numerous diseases including neurodegenerative disorders and certain cancers . ERdj3 antibodies are therefore valuable tools for investigating these fundamental cellular processes.

What are the key structural domains of ERdj3 and why are they important for antibody targeting?

ERdj3 contains several distinct structural domains that serve different functions and provide potential epitopes for antibody recognition:

• J-domain: Contains the critical HPD motif that mediates functional interactions with BiP. Mutations in this domain (such as H53Q) disrupt ERdj3-BiP interactions .

• Domain II: Contributes to ERdj3 tetramerization, with specific β-sheet structures (residues 175-190) being particularly important .

• Domain III: Essential for oligomerization, with the F326D mutation known to disrupt this process .

When generating or selecting antibodies against ERdj3, researchers should consider which domain they wish to target based on their experimental objectives. Antibodies targeting the J-domain may be useful for studying ERdj3-BiP interactions, while antibodies against domains involved in substrate binding might help investigate chaperone-substrate relationships. Understanding these structural features enables more precise epitope mapping and interpretation of antibody-based experimental results.

How does ERdj3 function in protein quality control pathways?

ERdj3 plays a multifaceted role in protein quality control within the ER:

• Initial substrate binding: Research indicates that ERdj3 may bind first to unfolded substrates and serve to inhibit protein aggregation until BiP joins the complex .

• Co-chaperone function: ERdj3 stimulates BiP's ATPase activity, enhancing BiP's ability to properly fold client proteins .

• Direct chaperone activity: Beyond its co-chaperone role, ERdj3 can directly bind to unfolded proteins and prevent their aggregation, demonstrating intrinsic chaperone activity .

• Substrate transfer mechanism: Current models suggest that ERdj3 initially binds unfolded proteins, recruits BiP to the complex, and then disassociates while BiP remains bound until folding is complete .

• Extracellular proteostasis: Uniquely, ERdj3 can be secreted from cells during ER stress, where it continues to bind misfolded proteins and prevent their extracellular aggregation and proteotoxicity .

This complex functionality makes ERdj3 antibodies particularly valuable for studying various aspects of cellular proteostasis both within the ER and in the extracellular environment.

What are the primary applications for ERdj3 antibodies in cellular research?

ERdj3 antibodies serve multiple critical functions in cellular research:

These applications enable researchers to investigate ERdj3's roles in protein folding, ER stress responses, and extracellular proteostasis regulation. When selecting antibodies for these applications, researchers should consider specificity, reactivity across species of interest, and validated performance in the specific application required .

How can I optimize co-immunoprecipitation protocols to study ERdj3-substrate interactions?

Optimizing co-immunoprecipitation (co-IP) protocols for ERdj3-substrate interactions requires careful consideration of several factors:

• Lysis buffer selection: Use mild detergents (such as 1% Triton X-100 or CHAPS) to preserve protein-protein interactions. Add protease inhibitors to prevent degradation during sample preparation.

• Crosslinking consideration: For transient interactions, such as those between ERdj3 and its substrates, chemical crosslinking (using DSP or formaldehyde) prior to lysis may help stabilize complexes. Research has shown that ERdj3 quickly disassociates from substrates before protein folding is completed .

• Antibody orientation: Consider whether to immunoprecipitate with anti-ERdj3 antibodies to pull down substrates, or with antibodies against the substrate to co-IP ERdj3. Each approach provides different insights.

• ATP/ADP manipulation: Since ERdj3-BiP interactions are influenced by nucleotide binding state, manipulating ATP/ADP levels in your buffers can affect complex stability. This is particularly relevant when studying the release of ERdj3 from substrate:BiP complexes .

• Control experiments: Include appropriate controls such as:

  • IgG control to assess non-specific binding

  • ERdj3 mutants (e.g., J-domain mutant H53Q) that cannot interact with BiP but retain substrate binding capability

  • Lysates from cells not expressing the substrate of interest

Following these optimization steps can significantly improve the detection of authentic ERdj3-substrate interactions while minimizing background.

What methodological considerations are important when using ERdj3 antibodies for immunofluorescence?

When using ERdj3 antibodies for immunofluorescence microscopy, several methodological considerations can enhance experimental success:

• Fixation method selection: For ER proteins like ERdj3, paraformaldehyde (4%) fixation often works well, but test different fixatives as they can affect epitope accessibility. Some epitopes may require methanol fixation for optimal detection.

• Permeabilization optimization: Since ERdj3 is a luminal ER protein, ensure adequate permeabilization (using Triton X-100, saponin, or digitonin) to allow antibody access to the ER lumen without disrupting ER morphology.

• Blocking conditions: Use 5-10% normal serum from the species in which the secondary antibody was raised, plus 1% BSA to reduce background staining.

• Co-staining considerations: When co-staining with other ER markers (e.g., BiP, calnexin), ensure antibodies are raised in different species to avoid cross-reactivity. This is particularly useful when investigating whether ERdj3 colocalizes with specific substrates or other chaperones .

• Controls for specificity:

  • Include a peptide competition assay using the immunizing peptide

  • Use siRNA knockdown of ERdj3 to confirm specificity

  • Include cells transfected with ERdj3 mutants as additional controls

• Secreted ERdj3 detection: For studies focusing on secreted ERdj3, consider using cellular secretion assays with supernatant collection and fixation of extracellular proteins onto coverslips before antibody incubation .

Following these methodological considerations will help ensure reliable and interpretable immunofluorescence results when studying ERdj3 localization and interactions.

How can ERdj3 antibodies be used to investigate the unfolded protein response (UPR)?

ERdj3 antibodies provide powerful tools for investigating various aspects of the unfolded protein response (UPR), a cellular stress pathway activated by ER stress:

• Expression level monitoring: During UPR activation, ERdj3 expression increases as part of the cell's adaptive response. Western blotting with validated ERdj3 antibodies can quantitatively track this upregulation in different cell types and under various stress conditions .

• Subcellular fractionation studies: Combining fractionation techniques with immunoblotting allows researchers to monitor potential redistribution of ERdj3 between ER, Golgi, and secretory vesicles during UPR activation.

• Secretion analysis: A distinctive feature of ERdj3 is its increased secretion during ER stress. ELISA or western blotting of concentrated cell culture media using ERdj3 antibodies can quantify this secretion, providing a unique biomarker of ER stress intensity and duration .

• Substrate protection assays: ERdj3 secreted during UPR can protect the extracellular environment from toxic protein conformations. Antibodies can help track this protective mechanism by immunoprecipitating ERdj3-substrate complexes from extracellular fluids .

• Co-secretion analysis: ERdj3 can be co-secreted with destabilized, misfolding-prone client proteins during ER stress. Immunoprecipitation with antibodies against either ERdj3 or the client protein can detect these complexes and help understand how the UPR links ER and extracellular proteostasis .

These approaches collectively provide a comprehensive view of how ERdj3 functions within the UPR network and extends proteostasis protection beyond the ER compartment.

What techniques allow investigation of ERdj3's direct binding to unfolded substrates versus BiP-mediated interactions?

Investigating the distinction between ERdj3's direct substrate binding versus BiP-mediated interactions requires sophisticated experimental approaches:

• Mutant ERdj3 analysis: Utilize ERdj3 mutants that selectively disrupt BiP binding (e.g., H53Q J-domain mutant) while retaining substrate binding capabilities. Research has shown that these mutants can still interact with unfolded protein substrates despite their inability to stimulate BiP's ATPase activity or bind BiP directly .

• Sequential immunoprecipitation: Perform initial immunoprecipitation with antibodies against substrate proteins, followed by dissociation of complexes and re-immunoprecipitation with either anti-ERdj3 or anti-BiP antibodies to determine the composition of chaperone complexes.

• In vitro binding assays: Using recombinant ERdj3 and denatured model substrates (such as RNAse A or Aβ peptides), assess direct binding in the absence of BiP through filter binding assays or pull-downs with ERdj3 antibodies .

• Temporal analysis of complex formation: Pulse-chase experiments combined with sequential immunoprecipitation can reveal the timing of ERdj3 and BiP associations with newly synthesized substrates. Research indicates that ERdj3 interacts with unfolded light chains initially but quickly dissociates before protein folding is completed, while BiP remains bound longer .

• ATP/ADP manipulation: Since BiP binding to substrates is ATP-dependent, manipulating nucleotide levels can help distinguish direct ERdj3-substrate interactions from BiP-dependent ones.

These techniques have revealed that ERdj3 binds directly to proteins rather than via interactions with BiP, supporting a model where ERdj3 may bind first to substrates and inhibit protein aggregation until BiP joins the complex .

How can researchers study the oligomerization state of ERdj3 and its impact on chaperone function?

Studying ERdj3 oligomerization and its functional implications requires specialized techniques:

• Gel filtration chromatography: This technique separates proteins based on size and can distinguish between monomeric, dimeric, and tetrameric states of ERdj3. Research has used this method to analyze the oligomeric state of wild-type ERdj3 and various mutants secreted from mammalian cells .

• Domain-specific mutant analysis: Generate and analyze specific ERdj3 mutants that affect oligomerization:

  • Domain II mutants (complete deletion or targeted deletion of residues 175-190)

  • Point mutations disrupting β-sheets (T177P, Q186P)

  • Domain III mutants (complete deletion or F326D point mutation)

• Chemical crosslinking: This approach can "freeze" oligomeric states for analysis by SDS-PAGE and western blotting with ERdj3 antibodies, revealing the distribution of different oligomeric species under various conditions.

• Functional correlation studies: Combine oligomerization analysis with functional assays (e.g., prevention of substrate aggregation, ATPase stimulation) to correlate oligomeric state with specific functions.

• Microscopy-based approaches: Techniques such as Förster resonance energy transfer (FRET) or fluorescence correlation spectroscopy (FCS) using fluorescently-tagged ERdj3 variants can provide insights into oligomerization dynamics in living cells.

Research has demonstrated that domain II plays a critical role in ERdj3 tetramerization, with specific β-sheet structures being particularly important. Domain III is also essential for oligomerization, with the F326D mutation disrupting this process . Understanding these structural determinants of oligomerization provides insight into how ERdj3's quaternary structure influences its chaperone function.

What are common challenges when using ERdj3 antibodies in western blotting and how can they be addressed?

Researchers commonly encounter several challenges when using ERdj3 antibodies for western blotting:

• Variable detection sensitivity: ERdj3 expression levels can change dramatically during ER stress conditions. Solution: Include positive controls from cells treated with known ER stress inducers (e.g., tunicamycin, thapsigargin) to establish detection sensitivity range.

• Multiple bands or unexpected molecular weight: ERdj3 undergoes N-glycosylation and possible post-translational modifications. Solution: Include deglycosylation controls (PNGase F treatment) to confirm band identity. Also consider that ERdj3's calculated molecular weight is approximately 40-43 kDa, but it may migrate differently on SDS-PAGE .

• Cross-reactivity with other DnaJ proteins: The J-domain is conserved across the DnaJ family. Solution: Validate antibody specificity using overexpression and knockdown controls. Consider using antibodies targeting less conserved regions of ERdj3.

• Detection of secreted versus intracellular ERdj3: Both pools may have different post-translational modifications. Solution: Separately analyze cell lysates and concentrated culture media, with appropriate loading controls for each fraction .

• Sample preparation issues: Improper lysis can affect protein extraction. Solution: Use ER-appropriate lysis buffers containing 1% Triton X-100 or CHAPS with complete protease inhibitors. For membrane-associated pools, consider using stronger detergents like SDS.

• Antibody dilution optimization: Polyclonal antibodies may require different optimization than monoclonals. Solution: Perform dilution series (typically 1:500 to 1:5000) to determine optimal signal-to-noise ratio for each specific antibody and application.

Addressing these challenges will help ensure reliable and reproducible western blotting results when studying ERdj3 expression and modifications.

How can researchers distinguish between wild-type ERdj3 and mutant variants in experimental settings?

Distinguishing between wild-type ERdj3 and various mutant variants requires strategic experimental approaches:

• Epitope tag differentiation: Engineer different epitope tags (HA, FLAG, Myc) on wild-type and mutant ERdj3 constructs to distinguish them using tag-specific antibodies. This approach was successfully used in research to differentiate wild-type ERdj3 from H53Q and other mutants .

• Mobility shift detection: Some mutations cause detectable shifts in electrophoretic mobility. For example, research has shown that the Q186P mutation produces a band with reduced mobility on SDS-PAGE compared to wild-type ERdj3 .

• Domain-specific antibodies: Generate or obtain antibodies that specifically recognize domains affected by mutations. For example, antibodies targeting the J-domain could differentially detect wild-type versus H53Q mutant.

• Functional assays: Use biochemical assays that reflect the functional differences:

  • BiP binding assays distinguish wild-type from H53Q mutant

  • Oligomerization assays differentiate wild-type from domain II/III mutants

  • Substrate binding persistence distinguishes wild-type (transient binding) from mutants with altered substrate release kinetics

• Immunoprecipitation patterns: Wild-type and mutant ERdj3 show different patterns of association with substrates and BiP. For instance, the ERdj3 H53Q mutant shows increased binding to immunoglobulin heavy chains compared to wild-type .

These approaches can be combined to create robust experimental systems for investigating how specific mutations affect ERdj3 structure and function in both cellular and biochemical contexts.

What controls are essential when studying ERdj3-substrate interactions?

When studying ERdj3-substrate interactions, several essential controls ensure experimental validity and data reliability:

• Negative interaction controls:

  • Non-client proteins that don't require chaperone assistance

  • IgG isotype control for immunoprecipitation experiments

  • Properly folded versions of the substrate being studied

• Functional mutant controls:

  • ERdj3 H53Q mutant: Disrupts BiP interaction but retains substrate binding capability

  • Substrate binding-deficient mutants of ERdj3

  • BiP binding-deficient mutants of substrate proteins

• Competition controls:

  • Excess unlabeled substrate in binding assays

  • Peptide competition for antibody specificity validation

  • ATP/ADP manipulation to distinguish nucleotide-dependent interactions

• Temporal controls:

  • Time course experiments to capture the transient nature of ERdj3-substrate interactions

  • Pulse-chase studies to track the sequential binding and release of ERdj3 and BiP

• Stress condition controls:

  • ER stress inducers (tunicamycin, thapsigargin) versus normal conditions

  • Heat shock versus normal temperature (for temperature-sensitive substrates)

• Binding specificity controls:

  • Denatured versus native substrate conformations

  • Various concentrations of denaturants to test binding stringency

  • Recombinant ERdj3 and various model substrates in vitro

Implementing these controls provides a comprehensive framework for validating the specificity, dynamics, and functional significance of ERdj3-substrate interactions in both cellular and biochemical experimental contexts.

How can ERdj3 antibodies be used to study extracellular proteostasis regulation?

The discovery that ERdj3 can be secreted and function in the extracellular environment opens new research avenues where antibodies play crucial roles:

• Secretion mechanism investigation: Use ERdj3 antibodies to examine the trafficking pathway of ERdj3 from the ER to secretory vesicles through immunofluorescence co-localization with markers of the secretory pathway.

• Quantitative secretion analysis: Develop sandwich ELISA assays with ERdj3 antibodies to quantify secreted ERdj3 levels in cell culture supernatants, tissue fluids, or patient samples under various physiological and pathological conditions .

• Extracellular complex identification:

  • Immunoprecipitate secreted ERdj3 from conditioned media to identify associated client proteins

  • Use antibodies against known amyloidogenic proteins (e.g., Aβ peptides) to co-immunoprecipitate bound ERdj3

  • Apply these techniques to study how ERdj3 prevents extracellular protein aggregation and proteotoxicity

• Functional intervention studies:

  • Use ERdj3 antibodies to neutralize secreted ERdj3 function in culture media

  • Apply antibodies to block specific domains of ERdj3 to determine their importance for extracellular chaperone function

  • Develop in vivo studies using ERdj3 antibodies to modulate extracellular proteostasis in disease models

• Co-secretion mechanisms: Investigate how ERdj3 co-secretes with misfolding-prone client proteins using dual immunofluorescence or proximity ligation assays with antibodies against both ERdj3 and client proteins .

Research has demonstrated that secreted ERdj3 can effectively inhibit the aggregation of amyloidogenic Aβ 1-40 peptide at substoichiometric concentrations, suggesting a potential role in preventing extracellular protein aggregation associated with neurodegenerative diseases .

What methods can be used to study the role of ERdj3 in disease models?

Investigating ERdj3's role in disease models requires integrated approaches where antibodies serve as critical tools:

• Expression profiling in disease tissues:

  • Immunohistochemistry with ERdj3 antibodies on patient-derived tissues

  • Western blot analysis of ERdj3 levels in disease models

  • Comparison of intracellular versus secreted ERdj3 pools in disease states

• Genetic manipulation approaches:

  • CRISPR/Cas9-mediated ERdj3 knockout or knock-in of disease-associated variants

  • siRNA knockdown followed by antibody-based detection of effects on client protein folding

  • Overexpression of wild-type or mutant ERdj3 with subsequent analysis of disease markers

• Patient-derived systems:

  • iPSC-derived cell types relevant to specific diseases

  • Analysis of ERdj3 function in primary cells from patients with protein folding diseases

  • Correlation of ERdj3 levels/function with disease severity using quantitative antibody-based assays

• Therapeutic potential assessment:

  • Administration of recombinant ERdj3 to disease models followed by antibody-based detection of effects on protein aggregation

  • Development of antibodies that enhance ERdj3 chaperone function

  • Screening for small molecules that modulate ERdj3 secretion or chaperone activity

• Amyloid disease model applications:

  • Use ERdj3 antibodies to examine colocalization with amyloid deposits in animal models

  • Investigate ERdj3 binding to Aβ in various aggregation states using co-immunoprecipitation

  • Assess the protective effect of secreted ERdj3 against Aβ toxicity

Research has shown that recombinant ERdj3 can completely inhibit the aggregation of Aβ 1-40 at concentrations above 370 nM (an ERdj3:Aβ 1-40 ratio of 1:27), suggesting therapeutic potential in amyloid-related disorders .

How does one interpret complex formation data between ERdj3, BiP, and substrate proteins?

Interpreting complex formation data between ERdj3, BiP, and substrate proteins requires consideration of several factors:

• Sequential binding kinetics:

  • ERdj3 typically binds unfolded substrates first, followed by BiP recruitment

  • ERdj3 dissociates relatively quickly, while BiP remains bound until folding is complete

  • Time course experiments must be interpreted with this sequential model in mind

• Ratio analysis considerations:

  • Substoichiometric amounts of ERdj3 can bind multiple substrate molecules

  • The ERdj3:BiP:substrate ratio may vary depending on substrate properties and folding stage

  • Quantitative western blotting or mass spectrometry can help determine these ratios

• Mutant comparison interpretation:

  • ERdj3 H53Q mutant shows increased binding to substrates due to inability to recruit BiP and trigger release

  • This suggests that interactions with BiP help trigger the release of ERdj3 from the substrate:BiP complex

  • Mutations affecting oligomerization may alter substrate binding patterns

• Differential co-immunoprecipitation patterns:

  • Co-IP from the substrate perspective versus from the chaperone perspective may yield different results

  • BiP-mediated release of ERdj3 means that BiP immunoprecipitation may not always pull down ERdj3

  • ERdj3 immunoprecipitation may capture only early folding intermediates

• Stress response adaptation:

  • During ER stress, increased ERdj3 expression alters the relative abundance of chaperone components

  • This may shift complex composition and stability in ways that require careful interpretation

  • Changes in secreted versus intracellular pools further complicate analysis

Understanding these complex dynamics helps researchers interpret experimental data correctly and develop more accurate models of how the ERdj3-BiP chaperone system functions in protein folding and quality control.