ERDJ3A Antibody

What is ERdj3 and why are antibodies against it important in research?

ERdj3 is an ER luminal protein and one of seven ER J-domain-containing Hsp40 co-chaperones. It serves critical functions in protein quality control by binding directly to unfolded client proteins and recruiting them to BiP for ATP-dependent chaperoning in the Hsp70-Hsp40-nucleotide exchange factor (NEF) folding cycle . Antibodies against ERdj3 are essential research tools that enable detection, quantification, and characterization of this protein in various experimental contexts, including immunoprecipitation, Western blotting, immunofluorescence, and proteomic studies.

The importance of these antibodies is highlighted by ERdj3's roles in multiple cellular processes including:

• Facilitating protein folding in the ER

• Participating in ER-associated degradation (ERAD) decisions

• Responding to ER stress conditions

• Contributing to disease mechanisms, such as in Gaucher's disease

Which epitopes of ERdj3 are most suitable for antibody targeting?

When selecting or generating antibodies against ERdj3, researchers should consider its domain structure. ERdj3 has 358 amino acids with a predicted size of 40.5 kDa and contains several distinct domains :

• N-terminal signal sequence (cleaved with 53% probability)

• J-domain (critical for BiP interaction)

• Substrate binding regions

• Dimerization domains

What controls should be included when validating ERdj3 antibodies?

Proper validation of ERdj3 antibodies requires several essential controls:

• Specificity controls:

  • Use of ERdj3 knockdown or knockout cells (ERdj3 shRNA has been successfully used in HEK293T-Rex cells)

  • Comparison with recombinant ERdj3 proteins (wild-type and mutant variants)

  • Pre-absorption with purified antigen

• Expression controls:

  • ER stress induction (e.g., thapsigargin treatment increases ERdj3 expression)

  • Comparison between intracellular and secreted forms

• Technical controls:

  • Use of tagged ERdj3 versions (HA-tagged variants have been successfully employed)

  • Molecular weight verification (cleaved signal sequence affects size)

A particularly useful approach involves comparing antibody reactivity between endogenous ERdj3 and exogenously expressed wild-type or mutant variants, which allows confirmation of specificity and characterization of epitope requirements .

How can ERdj3 antibodies distinguish between different conformational states?

ERdj3 exists in multiple conformational and functional states that researchers may wish to distinguish:

• Monomeric vs. dimeric forms

• BiP-bound vs. unbound states

• Substrate-bound vs. free states

• ER-resident vs. secreted forms

While standard polyclonal antibodies may not differentiate these states, specialized approaches can help:

• Conformation-specific antibodies: Raising antibodies against purified ERdj3 in specific conformational states

• Co-immunoprecipitation approaches: Using ERdj3 antibodies in native conditions to preserve protein-protein interactions

• Proximity ligation assays: Combining ERdj3 antibodies with antibodies against interaction partners

• Subcellular fractionation: Distinguishing ER-localized from secreted ERdj3

Research has demonstrated that ERdj3 forms dimers, and mixing tagged and untagged variants followed by immunoprecipitation with tag-specific antibodies can effectively study this phenomenon . Similarly, stress-induced ERdj3 secretion can be monitored by comparing intracellular and extracellular fractions using appropriate antibodies .

How can ERdj3 antibodies be used to study its role in protein quality control decisions?

ERdj3 plays a crucial role in determining whether misfolded proteins undergo further folding attempts or are targeted for degradation. Antibodies can help elucidate these mechanisms through several approaches:

• Sequential immunoprecipitation studies:

  • First precipitating substrate proteins (e.g., mutant GCase in Gaucher's disease)

  • Then detecting associated ERdj3 using specific antibodies

  • Quantifying the association over time to track quality control decisions

• Pulse-chase experiments with immunoprecipitation:

  • Metabolic labeling of cells with 35S-methionine and cysteine

  • Chasing in complete media for various times

  • Immunoprecipitation with ERdj3 antibodies to track association with substrate proteins over time

• BiP-ERdj3-substrate complex analysis:

  • Using ERdj3 antibodies alongside BiP antibodies to characterize the temporal dynamics of chaperone-substrate interactions

  • Comparing wild-type and mutant substrate proteins to understand quality control decision points

Research has shown that ERdj3 binds to unfolded substrates initially but disassociates before protein folding is completed, while BiP remains bound until folding is complete. This temporal dynamic is critical for understanding how ER quality control functions .

What methodological approaches best reveal ERdj3's interactions with the ERAD machinery?

ERdj3 contributes to ER-associated degradation (ERAD) of misfolded proteins, particularly in Gaucher's disease where mutations compromise β-glucocerebrosidase (GCase) folding. To study these interactions:

• Differential co-immunoprecipitation:

  • Use ERdj3 antibodies to immunoprecipitate under different conditions

  • Follow with mass spectrometry to identify ERAD components

  • Compare composition between wild-type and disease models

• ERdj3 depletion studies:

  • Measure degradation rates of ERAD substrates in ERdj3-depleted cells

  • Restore function with wild-type or mutant ERdj3 variants

  • Track changes in substrate localization and function

• Interaction mapping with key ERAD components:

  • Use proximity labeling approaches with ERdj3 antibodies

  • Study co-localization with Sec61 translocon and other ERAD machinery

Table of ERdj3-interacting proteins identified by GCase immunoprecipitation/mass spectrometry:

Evidence shows that depleting ERdj3 reduces the rate of mutant GCase degradation in patient-derived fibroblasts while increasing folding, trafficking, and function by directing GCase to the pro-folding ER calnexin pathway .

How can ERdj3 antibodies be used to investigate its role in ER stress responses?

ERdj3 is stress-inducible and contributes to the unfolded protein response (UPR). Antibodies can help characterize these functions through:

• Stress-dependent secretion analysis:

  • Compare intracellular and secreted ERdj3 levels under normal and stress conditions

  • Quantify the fold increase in secreted ERdj3 following stress induction

  • Correlate with activation of specific UPR branches

• UPR target monitoring:

  • Track ERdj3 expression alongside other UPR-regulated genes

  • Use quantitative immunoblotting to assess temporal dynamics during stress

  • Compare ERdj3 levels with BiP and other chaperones

• Subcellular redistribution studies:

  • Use immunofluorescence with ERdj3 antibodies to track localization during stress

  • Perform subcellular fractionation followed by immunoblotting

  • Analyze stress-dependent protein complexes via native gel electrophoresis

Research has demonstrated that ER stress significantly increases ERdj3 secretion, which can be measured by immunoblotting media samples or by pulse-chase experiments using metabolic labeling followed by immunoprecipitation with ERdj3 antibodies .

What approaches are most effective for studying ERdj3 mutations in disease models?

To investigate how ERdj3 mutations contribute to disease pathology:

• Comparative immunoprecipitation:

  • Use antibodies that recognize both wild-type and mutant ERdj3

  • Compare substrate binding profiles between variants

  • Assess changes in BiP recruitment efficiency

• Structure-function analysis:

  • Generate domain-specific antibodies or epitope-tagged domain mutants

  • Correlate structural features with functional outcomes

  • Study the impact of mutations on ERdj3 dimerization

• Disease-relevant substrate interactions:

  • In Gaucher's disease models, track ERdj3-GCase interactions

  • In models of ER stress-related diseases, monitor ERdj3 secretion

  • In protein misfolding diseases, assess changes in quality control decisions

Particularly revealing are experiments with the ERdj3 QPD mutant (in which the HPD motif is mutated to QPD), which abolishes BiP binding but preserves substrate interactions. Studies have shown this mutant binds substrates more stably than wild-type ERdj3, providing insights into the mechanism by which BiP interaction triggers ERdj3 release from substrates .

How can researchers address inconsistent ERdj3 detection across different cell types?

Researchers may encounter variability in ERdj3 detection across different cell types or experimental conditions. This may result from:

• Expression level differences:

  • ERdj3 expression varies by cell type and stress conditions

  • Baseline expression may be low in some cells but dramatically increase under ER stress

  • Recommendation: Include positive controls with known ERdj3 expression and compare results under both normal and stressed conditions

• Complex formation interference:

  • ERdj3 exists predominantly in protein complexes rather than as a free pool

  • These interactions may mask antibody epitopes

  • Recommendation: Use sample preparation methods that preserve or disrupt complexes as needed, and try multiple antibodies targeting different epitopes

• Topology and post-translational modification differences:

  • ERdj3's topology can affect antibody accessibility

  • Signal sequence cleavage affects molecular weight

  • Recommendation: Verify the expected molecular weight for the specific cell type and confirm with tagged recombinant proteins as size references

What are the optimal conditions for immunoprecipitating ERdj3-substrate complexes?

The transient nature of ERdj3's interactions with client proteins can complicate immunoprecipitation experiments. Consider these approaches:

• Stabilizing complexes:

  • Use chemical crosslinkers to capture transient interactions

  • Employ ATP depletion to stabilize chaperone-substrate complexes

  • Consider co-expression of ERdj3 QPD mutant which forms more stable substrate interactions

• Buffer optimization:

  • Avoid harsh detergents that may disrupt protein-protein interactions

  • Include ATP or ATP analogs as appropriate for the specific complex

  • Control buffer pH and salt concentration to preserve native interactions

• Sequential immunoprecipitation strategy:

  • First precipitate the substrate protein

  • Then detect ERdj3 in the precipitate

  • Alternatively, first precipitate ERdj3 and then detect substrates

Researchers have successfully used these approaches to study ERdj3 interactions with immunoglobulin heavy chains, light chains, and other substrates, demonstrating that careful optimization can preserve these biologically relevant complexes .

How can researchers differentiate between monomeric and dimeric forms of ERdj3?

ERdj3 forms multimers in cells, which can complicate antibody-based analyses. To distinguish these forms:

• Native gel electrophoresis:

  • Run samples under non-denaturing conditions

  • Use ERdj3 antibodies for Western blotting

  • Compare migration patterns with known molecular weight standards

• Co-expression of tagged variants:

  • Express both HA-tagged and untagged ERdj3 simultaneously

  • Immunoprecipitate with anti-HA antibodies

  • Detect untagged ERdj3 co-precipitation to confirm dimerization

• Size exclusion chromatography:

  • Fractionate cell lysates by molecular size

  • Use ERdj3 antibodies to identify which fractions contain the protein

  • Compare with standards to determine oligomeric state

Research has demonstrated that wild-type ERdj3 and the QPD mutant readily form mixed dimers when co-expressed, suggesting that J-domain mutations do not affect dimerization capability .

How might antibodies help elucidate ERdj3's dual topology in the ER membrane?

Recent research suggests ERdj3 may have a dual topology at the ER membrane rather than being exclusively luminal:

• Topology-specific antibodies:

  • Generate antibodies against domains predicted to have different accessibility

  • Use selective permeabilization in immunofluorescence

  • Compare antibody reactivity in intact versus disrupted membranes

• Protease protection assays:

  • Combine with domain-specific antibodies

  • Compare accessibility of different regions to proteolytic digestion

  • Correlate findings with computational predictions of transmembrane domains

• Proximity labeling approaches:

  • Fuse BioID or APEX2 to ERdj3 domains

  • Identify neighboring proteins using mass spectrometry

  • Determine which cellular compartments contain ERdj3-interacting proteins

Computational analysis predicts a signal peptide and two transmembrane domains in ERdj3, with the first transmembrane domain being located within the signal sequence. Further research using topology-specific antibodies could help resolve the conflicting data regarding ERdj3's membrane association and orientation .

What is the potential for ERdj3 antibodies in studying stress-induced secretion mechanisms?

The discovery that ERdj3 can be secreted under ER stress conditions opens new research avenues:

• Secretion pathway characterization:

  • Use antibodies to track ERdj3 through the secretory pathway

  • Determine if secreted ERdj3 retains chaperone activity

  • Investigate whether secreted ERdj3 serves extracellular functions

• Quantification of stress-induced secretion:

  • Develop sensitive ELISAs using ERdj3 antibodies

  • Compare secretion levels under different stress conditions

  • Correlate with other UPR markers to identify regulatory mechanisms

• Therapeutic implications:

  • Determine if secreted ERdj3 levels correlate with disease states

  • Investigate the potential of secreted ERdj3 as a biomarker

  • Explore whether antibodies targeting secreted ERdj3 could have therapeutic value

Research has shown that ER stress significantly increases both ERdj3 expression and secretion. Quantitative analysis of media and cell lysates has demonstrated up to 3-fold increases in secreted ERdj3 following thapsigargin treatment, suggesting this process is regulated as part of the cellular stress response .