DNAJA1 Antibody

What is DNAJA1 and what cellular functions does it perform?

DNAJA1 (DnaJ homolog subfamily A member 1) is a member of the DnaJ protein family (also known as Hsp40 or Hsc40) that functions as a cochaperone to Hsp70 proteins. It has a calculated molecular weight of 45 kDa (397 amino acids) and is widely expressed across human, mouse, and rat tissues .

DNAJA1 serves multiple critical cellular functions:

• Stimulates ATP hydrolysis in Hsp70 chaperone systems

• Facilitates protein transport into mitochondria through its co-chaperone activity

• Acts as a negative regulator of BAX translocation from cytosol to mitochondria under cellular stress conditions, providing protection against apoptosis

• Can promote apoptosis in response to specific cellular stressors like anisomycin or UV exposure

• Plays a significant role in protein triage decisions, particularly for proteins involved in neurodegenerative diseases such as tau

The cellular functions of DNAJA1 are context-dependent, with its role in protein triage and apoptosis regulation being particularly relevant to neurodegenerative disease research.

Which experimental applications are validated for DNAJA1 antibodies?

DNAJA1 antibodies have been validated for multiple experimental applications across various sample types. The comprehensive validation data shows reproducible results across several experimental contexts.

Note: For IHC applications, antigen retrieval with TE buffer pH 9.0 is suggested, with citrate buffer pH 6.0 as an alternative option .

How should researchers optimize Western blot protocols for DNAJA1 detection?

Optimizing Western blot protocols for DNAJA1 detection requires attention to several critical parameters:

• Sample preparation: When lysing cells or tissues, include protease inhibitors in your lysis buffer to prevent DNAJA1 degradation. The protein is well-detected in multiple sample types including cell lines (HEK293, HeLa, HepG2, Jurkat) and tissue samples (human liver, testis, mouse/rat kidney) .

• Antibody selection and dilution: Commercial antibodies like 11713-1-AP (polyclonal) have been validated at dilutions of 1:1000-1:8000 for Western blot. Monoclonal antibodies like EPR7248 (ab126774) show high specificity at 1:1000 dilution .

• Controls: Include appropriate positive controls such as HepG2 or Jurkat cell lysates, which consistently show DNAJA1 expression. For negative controls, DNAJA1 knockout cell lysates (like DNAJA1 KO HEK293T) provide excellent specificity verification .

• Detection of expected band: The predicted molecular weight of DNAJA1 is 45 kDa, but it often appears at around 49 kDa in SDS-PAGE. This difference between predicted and observed size should be considered when evaluating results .

• Loading control selection: GAPDH (36 kDa) or Vinculin (130 kDa) have been successfully used as loading controls in DNAJA1 detection systems .

Each antibody should be titrated in your specific experimental system to achieve optimal signal-to-noise ratio, as the optimal dilution may vary depending on the sample type and detection method.

What are the recommended approaches for immunofluorescence experiments with DNAJA1 antibodies?

For successful immunofluorescence experiments with DNAJA1 antibodies, researchers should implement the following methodological approach:

• Cell selection: HepG2 cells have been validated for positive DNAJA1 detection by immunofluorescence and serve as an excellent model system .

• Fixation and permeabilization: Standard 4% paraformaldehyde fixation (10-15 minutes at room temperature) followed by permeabilization with 0.1-0.25% Triton X-100 works well for DNAJA1 detection.

• Antibody dilution: Use DNAJA1 antibodies at dilutions ranging from 1:50-1:500, with optimization recommended for each experimental system .

• Co-localization studies: DNAJA1 can be effectively co-stained with tau protein to study their interaction. In HeLa cells stably overexpressing tau, transiently transfected flag-tagged DNAJA1 has been successfully visualized together with tau using anti-Flag (green) and anti-tau (red) antibodies, with Hoechst counterstaining for nuclei .

• Subcellular localization observations: Pay particular attention to the peri-nuclear region when examining DNAJA1 and tau interactions, as inversely co-localized patterns with peri-nuclear tau aggregates have been observed in neurons from tau transgenic mice .

For flow cytometry applications with intracellular staining, a 1:10 dilution has been validated using Jurkat cells, with rabbit IgG serving as an appropriate negative control .

How does DNAJA1 influence tau protein stability and what are the implications for neurodegenerative disease research?

DNAJA1 plays a crucial role in tau protein triage with significant implications for neurodegenerative disease research. The relationship between DNAJA1 and tau stability is characterized by several key mechanisms:

• Inverse correlation between DNAJA1 and tau levels: Research demonstrates that DNAJA1 and tau levels are inversely correlated both in cells and brain tissue. When DNAJA1 levels are reduced, tau levels increase, and vice versa. Experimental overexpression of DNAJA1 has been shown to reduce tau levels by approximately 47%, while knockdown of DNAJA1 results in increased tau accumulation .

• Direct binding interaction: Immunoprecipitation experiments have confirmed that DNAJA1 directly binds to tau protein. This interaction influences the association of tau with Hsp70, as evidenced by increased binding of Hsp70 to tau in the presence of DNAJA1 overexpression .

• Tau triage for degradation: DNAJA1 functions to triage all tau species for ubiquitin-dependent clearance mechanisms. This function is dependent on the integrity of residues linked to ubiquitination in human disease .

• Counterbalance with Hsp70: The relationship between DNAJA1 and Hsp70 is competitive regarding tau stability. When Hsp70 levels are concomitantly increased with DNAJA1, the tau-reducing effects of DNAJA1 are abrogated. This suggests a critical balance between these two chaperones in determining tau fate .

• Implications for neurodegenerative diseases: In Alzheimer's disease and tau transgenic mouse models, the absence of DNAJA1 may be either a cause or effect of tau accumulation. This positions the DnaJ protein family as potentially powerful genetic modifiers for tau pathogenesis .

For researchers investigating neurodegenerative diseases, these findings suggest that DNAJA1 modulation could represent a therapeutic approach for reducing pathological tau accumulation. Experimental designs should consider the balance between DNAJA1 and Hsp70 levels, as this appears to be a critical determinant in tau triage decisions.

What experimental approaches can be used to study DNAJA1's role in protein triage mechanisms?

To investigate DNAJA1's role in protein triage mechanisms, researchers can employ several sophisticated experimental approaches:

• Genetic manipulation of DNAJA1 expression:

  • Overexpression using flag-tagged DNAJA1 constructs in cell models that express tau or other proteins of interest

  • Knockdown using siRNA or shRNA targeting DNAJA1

  • CRISPR/Cas9-mediated knockout, as demonstrated with the DNAJA1 knockout HEK-293T cell line (available as ab266437)

• Protein-protein interaction studies:

  • Co-immunoprecipitation experiments to detect direct interactions between DNAJA1, Hsp70, and client proteins

  • Proximity ligation assays to visualize protein interactions in situ

  • FRET or BiFC approaches to monitor dynamic interactions in living cells

• Ubiquitination and degradation pathway analysis:

  • Proteasome inhibitors (e.g., MG132) can be used to determine if DNAJA1-mediated protein degradation occurs via the ubiquitin-proteasome system

  • Ubiquitination assays to directly measure changes in client protein ubiquitination when DNAJA1 levels are manipulated

  • Pulse-chase experiments to measure protein half-life changes in response to DNAJA1 manipulation

• Client protein specificity assessment:

  • Compare effects of DNAJA1 on different proteins (e.g., tau, polyQ proteins, α-synuclein) to determine specificity

  • Domain mapping using truncated DNAJA1 constructs to identify regions responsible for specific client recognition

• Hsp70-DNAJA1 balance manipulation:

  • Co-expression experiments with varying ratios of DNAJA1 and Hsp70 to determine how their balance affects client protein fate

  • Use of Hsp70 mutants that cannot bind tau (e.g., mτ-Hsp70) to investigate dependency of DNAJA1 effects on Hsp70

Research has shown that DNAJA1 effects display some client specificity - it reduces levels of tau and polyQ proteins but has minimal effects on α-synuclein. This suggests that experimental designs should include multiple client proteins to determine the specificity of observed effects .

How do DNAJA1 and Hsp70 cooperatively regulate protein homeostasis in cellular stress responses?

DNAJA1 and Hsp70 engage in a complex regulatory relationship to maintain protein homeostasis during cellular stress, with several key mechanisms:

The experimental evidence indicates that any imbalance in Hsp/c70 and DNAJA1 levels could dramatically impact protein accumulation, particularly for disease-associated proteins like tau. This balance may represent a critical factor in neurodegenerative diseases where protein aggregation is a key pathological feature.

What technical considerations are important when validating DNAJA1 antibody specificity?

Validating DNAJA1 antibody specificity requires rigorous technical approaches to ensure reliable experimental results:

How should researchers design experiments to investigate DNAJA1's role in neurodegenerative disease models?

Designing rigorous experiments to investigate DNAJA1's role in neurodegenerative disease models requires careful consideration of several methodological aspects:

• Model system selection:

  • Cellular models: HeLa or M17 neuroblastoma cells expressing wild-type or mutant tau provide accessible systems for mechanistic studies

  • Primary neurons: More physiologically relevant but technically challenging; useful for confirming findings from cell lines

  • Transgenic mice: Tau transgenic mice allow for in vivo investigation of DNAJA1-tau interactions in the context of neurodegeneration

  • Human tissue: Post-mortem brain tissue from neurodegenerative disease patients provides validation in the disease context

• DNAJA1 manipulation strategies:

  • Overexpression: Use flag-tagged DNAJA1 constructs to track the protein while examining effects on tau or other disease-associated proteins

  • Knockdown/Knockout: siRNA, shRNA, or CRISPR approaches to reduce or eliminate DNAJA1 expression

  • Point mutations: Introduce mutations in key functional domains (e.g., J-domain, client-binding regions) to dissect mechanistic requirements

• Investigation of Hsp70-DNAJA1 balance:

  • Design co-expression experiments with varying ratios of DNAJA1 and Hsp70

  • Use Hsp70 mutants that cannot bind tau (mτ-Hsp70) to investigate dependency of DNAJA1 effects

  • Pharmacological manipulation of the Hsp70-DNAJA1 interaction using small molecules

• Readout measurements:

  • Protein levels: Western blot analysis of tau, phospho-tau, and other disease-relevant proteins

  • Protein aggregation: Filter trap assays, ThT fluorescence, or immunofluorescence visualization of aggregates

  • Neuronal health: Viability assays, neurite outgrowth, electrophysiology

  • Behavioral assessments: For in vivo models, cognitive and motor function tests

• Spatiotemporal considerations:

  • Examine DNAJA1 localization relative to protein aggregates

  • Track changes in DNAJA1 expression and localization during disease progression

  • Consider age-dependent changes in chaperone systems in models of age-related neurodegenerative diseases

• Translational relevance:

  • Correlate findings with human post-mortem tissue samples from patients with tauopathies or other neurodegenerative diseases

  • Examine genetic variations in DNAJA1 and their association with disease risk or progression

  • Consider therapeutic approaches based on modulating DNAJA1 activity or expression

Evidence indicates that in AD and tau transgenic mice, the absence of DNAJA1 could be a critical cause or effect of tau accumulation. Therefore, experimental designs should carefully assess both causality and correlation in the relationship between DNAJA1 levels and disease phenotypes .

What controls should be included when studying protein-protein interactions involving DNAJA1?

When investigating protein-protein interactions involving DNAJA1, a comprehensive set of controls is essential to ensure reliable and interpretable results:

• Input controls:

  • Expression level verification: Western blot analysis to confirm expression levels of DNAJA1 and potential interacting partners before interaction studies

  • Subcellular localization: Immunofluorescence to verify that both proteins localize to the same cellular compartments where interaction is being studied

• Co-immunoprecipitation controls:

  • IgG control: Non-specific IgG of the same species as the immunoprecipitating antibody

  • Reverse co-IP: Immunoprecipitate with antibodies against both DNAJA1 and the putative interacting protein

  • Bead-only control: Include a sample with beads but no antibody to detect non-specific binding

  • Input sample: Include a sample of the total lysate before immunoprecipitation (typically 5-10%)

• Specificity controls:

  • DNAJA1 knockout/knockdown cells: Essential negative control for validating interactions

  • Domain mutants: DNAJA1 constructs with mutations in key functional domains to map interaction surfaces

  • Competition assays: Use purified domains to compete with full-length protein interactions

• Functional validation controls:

  • Hsp70 co-expression: As DNAJA1 functions as a co-chaperone for Hsp70, varying Hsp70 levels can confirm functional relevance of interactions

  • ATP/ADP modulation: Since DNAJA1-Hsp70 interactions are nucleotide-dependent, including conditions with ATP, ADP, or non-hydrolyzable ATP analogs can provide mechanistic insights

• Context-dependent controls:

  • Stress conditions: Compare interactions under normal and stress conditions (heat shock, oxidative stress)

  • Post-translational modifications: Examine how phosphorylation or other modifications affect interactions

  • Cell type specificity: Verify interactions in multiple cell types relevant to the research question

• Technical controls for imaging-based interaction studies:

  • Antibody specificity: Secondary-only controls and peptide competition assays

  • Cross-bleed controls: When using multiple fluorophores, single-label controls to detect bleed-through

  • Colocalization quantification: Use appropriate statistical measures (Pearson's coefficient, Manders' overlap) rather than subjective assessment

Research has shown that DNAJA1 interacts with tau and influences its association with Hsp70. These interactions can be visualized through co-immunoprecipitation and co-localization experiments. The quality of these experiments depends critically on the inclusion of appropriate controls to distinguish specific from non-specific interactions .

What are common challenges in DNAJA1 detection and how can they be addressed?

Researchers frequently encounter several challenges when detecting DNAJA1 in experimental systems. Here are common problems and their methodological solutions:

• Multiple bands in Western blot:

  • Challenge: Appearance of additional bands besides the expected 45-49 kDa DNAJA1 band

  • Solution: Validate specificity using DNAJA1 knockout controls; optimize antibody concentration (typically 1:1000-1:8000 for WB); increase washing stringency; consider using monoclonal antibodies like EPR7248 which may offer higher specificity

• Variable expression levels across samples:

  • Challenge: Inconsistent DNAJA1 detection between experiments or sample types

  • Solution: Use validated positive controls (HepG2, Jurkat, or HEK293 cells); standardize sample preparation methods; ensure equal loading with reliable loading controls like GAPDH or Vinculin; consider that DNAJA1 expression may be stress-responsive

• Poor signal in immunohistochemistry:

  • Challenge: Weak or absent staining in tissue sections

  • Solution: Optimize antigen retrieval (try both TE buffer pH 9.0 and citrate buffer pH 6.0); adjust antibody concentration (1:50-1:500 is recommended range); extend primary antibody incubation time; use amplification systems if necessary

• Background in immunofluorescence:

  • Challenge: High background or non-specific staining

  • Solution: Increase blocking time and concentration; optimize antibody dilution (1:50-1:500 range); include additional washing steps; use fluorophore-conjugated secondary antibodies with minimal cross-reactivity

• Detection in co-immunoprecipitation experiments:

  • Challenge: Difficulty detecting DNAJA1 interactions with client proteins

  • Solution: Use mild lysis conditions to preserve protein-protein interactions; optimize salt and detergent concentrations; consider crosslinking approaches for transient interactions; ensure antibodies don't compete for the same binding region

• Species cross-reactivity issues:

  • Challenge: Antibody performs differently across human, mouse, and rat samples

  • Solution: Select antibodies validated for your species of interest; compare sequence homology in the epitope region; consider species-specific antibodies for critical experiments

• Protein degradation during sample preparation:

  • Challenge: Loss of DNAJA1 signal during extraction

  • Solution: Include protease inhibitors in lysis buffers; maintain samples at 4°C during processing; avoid freeze-thaw cycles; consider fresh samples over frozen when possible

• Flow cytometry optimization:

  • Challenge: Poor separation between positive and negative populations

  • Solution: Ensure thorough permeabilization for this intracellular protein; use higher antibody concentration (1:10 dilution has been validated); include proper compensation controls; use rabbit IgG as negative control

For all applications, proper antibody validation using positive and negative controls is essential for troubleshooting and optimizing DNAJA1 detection protocols.

How can researchers interpret contradictory results when studying DNAJA1's effects on protein stability?

When faced with contradictory results in studies of DNAJA1's effects on protein stability, researchers should consider several methodological and biological factors that might explain these discrepancies:

• Cell type-specific effects:

  • Different cell types may have varying baseline levels of Hsp70 family members, which critically influence DNAJA1 function

  • The ratio between DNAJA1 and Hsp70 appears to be a key determinant of client protein fate

  • Solution: Compare DNAJA1:Hsp70 ratios across experimental systems and standardize or account for these differences

• Client protein specificity:

  • DNAJA1 shows differential effects on various client proteins (strong effects on tau and polyQ proteins, minimal effects on α-synuclein)

  • Solution: Consider that contradictory results may reflect genuine biological differences in how DNAJA1 recognizes and processes different client proteins

• Experimental timeframes:

  • Acute vs. chronic manipulation of DNAJA1 levels may yield different results due to compensatory mechanisms

  • Solution: Include time-course experiments and consider both short-term and long-term readouts of protein stability

• Post-translational modifications:

  • Both DNAJA1 and its client proteins undergo various post-translational modifications that affect their interactions

  • Solution: Characterize the modification status of DNAJA1 and client proteins in your experimental system using phospho-specific antibodies or mass spectrometry

• Expression level artifacts:

  • Overexpression systems may create non-physiological interactions or overwhelming of regulatory systems

  • Solution: Use multiple expression levels and consider knock-in approaches for more physiological expression

• Cellular stress context:

  • DNAJA1 functions differently under various stress conditions

  • Solution: Standardize experimental conditions and consider that contradictory results may reflect differences in cellular stress status

• Methodological differences in protein stability measurement:

  • Protein stability can be measured through various approaches (half-life studies, steady-state levels, etc.)

  • Solution: Use multiple complementary approaches to measure protein stability and degradation rates

• Genetic background variations:

  • In animal models or cell lines, background genetic differences can influence chaperone network function

  • Solution: Use isogenic cell lines or backcrossed animal models to minimize background effects

A prime example of potentially contradictory results comes from the dual role of DNAJA1 in apoptosis regulation. Research has shown it can both protect cells against apoptosis (by negatively regulating BAX translocation) and promote apoptosis in response to specific stressors (anisomycin or UV exposure). These seemingly contradictory functions likely reflect context-dependent roles rather than experimental artifacts .

When DNAJA1 is co-expressed with Hsp70, its effect on tau stability is abrogated, which might appear contradictory to its tau-reducing effect when expressed alone. This apparent contradiction actually reveals the complex regulatory relationship between these chaperones .

What emerging technologies could advance our understanding of DNAJA1 function in protein quality control?

Several cutting-edge technologies offer promising approaches to deepen our understanding of DNAJA1's function in protein quality control systems:

• Proximity-based proteomics:

  • BioID or APEX2 fusion proteins can identify the complete interactome of DNAJA1 in living cells

  • TurboID variants provide temporal resolution to capture dynamic interactions during stress responses

  • These approaches could identify novel DNAJA1 clients and regulatory partners beyond currently known proteins like tau

• Cryo-electron microscopy:

  • High-resolution structural studies of DNAJA1 in complex with Hsp70 and client proteins

  • Visualization of conformational changes during the client protein triage process

  • Understanding the structural basis for client specificity (why DNAJA1 affects tau and polyQ proteins but not α-synuclein)

• Live-cell imaging of protein quality control:

  • FRET-based sensors to monitor DNAJA1-client interactions in real-time

  • Photoconvertible fluorescent protein fusions to track protein degradation rates

  • Super-resolution microscopy to visualize DNAJA1 localization within quality control compartments

• Single-cell technologies:

  • Single-cell proteomics to examine cell-to-cell variability in DNAJA1 function

  • Single-cell RNA-seq to correlate DNAJA1 expression with client protein levels

  • These approaches could reveal how cellular heterogeneity influences protein quality control decisions

• CRISPR-based screening approaches:

  • Genome-wide CRISPR screens to identify genetic modifiers of DNAJA1 function

  • CRISPRi/CRISPRa libraries to modulate expression of chaperone network components

  • Base editing to introduce specific mutations in DNAJA1 or client proteins

• Protein-specific degradation technologies:

  • Targeted protein degradation using PROTACs or dTAGs to achieve temporal control of DNAJA1 levels

  • Optogenetic control of DNAJA1 activity to manipulate protein quality control with spatial and temporal precision

  • These approaches could overcome limitations of traditional overexpression or knockout strategies

• In silico modeling approaches:

  • Molecular dynamics simulations of DNAJA1-client interactions

  • Systems biology models of the complete chaperone network

  • Machine learning approaches to predict DNAJA1 client specificity based on protein features

• Translational research technologies:

  • Patient-derived iPSCs differentiated into neurons to study DNAJA1 function in disease-relevant human cells

  • Humanized mouse models carrying patient-specific variants in DNAJA1 or interacting proteins

  • Small molecule screens to identify compounds that modulate DNAJA1-mediated protein triage

Given that DNAJA1 appears to affect specific client proteins differently (strong effects on tau and polyQ proteins, minimal effect on α-synuclein), technologies that can systematically define client specificity determinants would be particularly valuable for understanding its role in neurodegenerative diseases .

How might therapeutic approaches targeting DNAJA1 be developed for neurodegenerative diseases?

The development of therapeutic approaches targeting DNAJA1 for neurodegenerative diseases represents an emerging frontier with several promising strategies:

• Modulation of DNAJA1 expression levels:

  • Rationale: Research has shown an inverse correlation between DNAJA1 and tau levels, suggesting that increasing DNAJA1 expression could reduce pathological tau accumulation

  • Approaches: Gene therapy using AAV vectors to deliver DNAJA1; small molecules that induce DNAJA1 expression; microRNA modulators that target DNAJA1 regulators

  • Considerations: Tissue-specific and cell-type-specific delivery systems would be necessary to target neurons affected in specific neurodegenerative diseases

• Targeting DNAJA1-Hsp70 balance:

  • Rationale: The balance between DNAJA1 and Hsp70 appears critical for determining tau fate; when Hsp70 levels are concomitantly increased with DNAJA1, the tau-reducing effects of DNAJA1 are abrogated

  • Approaches: Develop compounds that specifically modulate the DNAJA1:Hsp70 ratio; target the DnaJ-binding domain of Hsp70 to enhance DNAJA1-mediated tau clearance

  • Considerations: Careful titration would be necessary to achieve beneficial effects without disrupting essential chaperone functions

• Client-specific targeting approaches:

  • Rationale: DNAJA1 shows differential effects on various client proteins (strong effects on tau and polyQ proteins, minimal effects on α-synuclein)

  • Approaches: Develop molecules that enhance DNAJA1 recognition of specific disease-associated proteins; design chimeric proteins that bring DNAJA1 in proximity to target proteins

  • Considerations: Understanding the molecular basis of client specificity is prerequisite for rational design of such approaches

• Post-translational modification modulators:

  • Rationale: DnaJ proteins may recognize discrete post-translationally modified species

  • Approaches: Develop compounds that modify DNAJA1 or client protein post-translational modifications to enhance recognition and clearance

  • Considerations: Requires detailed understanding of which modifications influence DNAJA1-client interactions

• Targeting DNAJA1's role in protein degradation pathways:

  • Rationale: DNAJA1 triages tau for ubiquitin-dependent clearance mechanisms

  • Approaches: Develop compounds that enhance DNAJA1-mediated client ubiquitination or targeting to degradation machinery

  • Considerations: May require combination approaches with proteasome or autophagy modulators

• Precision medicine approaches:

  • Rationale: Individual variations in the chaperone network may influence disease progression and therapeutic response

  • Approaches: Genetic screening for DNAJA1 variants or expression levels to stratify patients; personalized treatment based on chaperone network status

  • Considerations: Requires development of biomarkers for chaperone network function

• Combinatorial therapeutic strategies:

  • Rationale: The complex nature of protein homeostasis suggests multi-target approaches may be necessary

  • Approaches: Combine DNAJA1-targeting approaches with other proteostasis modulators or disease-specific therapeutics

  • Considerations: Careful evaluation of drug-drug interactions and synergistic/antagonistic effects

Research has demonstrated that in Alzheimer's disease and tau transgenic mice, the absence of DNAJA1 could be a critical cause or effect of tau accumulation. This positions DNAJA1 as a potential therapeutic target, but successful therapeutic development will require deeper understanding of its complex regulatory relationships with other components of the protein quality control system .