ATAD3A Antibody, HRP conjugated

What is the specific binding region of ATAD3A recognized by commonly available HRP-conjugated antibodies?

Most commercially available HRP-conjugated ATAD3A antibodies target amino acids 2-240 of the human ATAD3A protein . This N-terminal region contains important structural domains that are accessible for antibody binding. When designing experiments, it's important to consider that this binding specificity could affect detection of certain ATAD3A truncated variants. For instance, the ATAD3A ΔN50 mutant (lacking the first 50 amino acids) that has been implicated in enhanced oligomerization might show altered binding patterns with antibodies targeting this region .

What is the appropriate working dilution range for HRP-conjugated ATAD3A antibodies in different applications?

For ELISA applications, HRP-conjugated ATAD3A antibodies typically perform optimally at dilutions between 1:1000 to 1:5000, depending on the specific antibody and experimental conditions . When adapting these antibodies for Western blotting applications, researchers should start with a 1:500 dilution and optimize based on signal intensity and background levels. For studying ATAD3A oligomerization in neurodegenerative disease models, where detecting both monomeric and oligomeric forms is critical, titration experiments should be performed to ensure optimal detection of both species without signal saturation .

How should I design experiments to detect ATAD3A oligomerization using HRP-conjugated antibodies?

Based on established protocols, ATAD3A oligomerization can be detected using Western blotting under non-reducing conditions (absence of β-mercaptoethanol) . When designing such experiments:

• Sample preparation: Prepare protein lysates without reducing agents to preserve disulfide-mediated oligomeric structures

• Include positive controls: Samples from known oligomerization-inducing conditions (e.g., cells treated with oligomeric Aβ1-42 peptides)

• Comparative analysis: Run parallel samples under reducing conditions (with β-mercaptoethanol) to confirm the disulfide-dependent nature of the oligomers

• Molecular weight markers: Use appropriate markers that cover the range of both monomeric (~66 kDa) and oligomeric (primarily dimers at ~130 kDa) ATAD3A species

• Loading controls: Include mitochondrial markers like VDAC or Tom20 for normalization of mitochondrial protein content

For chemical cross-linking experiments to stabilize oligomers, bismaleimidohexane (BMH) can be used as demonstrated in APP wildtype and APP Swedish mutant-expressing cells .

What controls should be included when studying ATAD3A in Alzheimer's disease models?

When investigating ATAD3A in AD models, comprehensive controls should include:

• Tissue/cell type matched controls: Compare AD patient samples with age-matched control subjects or AD model mice (e.g., 5XFAD) with wildtype littermates

• Regional controls: Include both affected brain regions (cortex, hippocampus, thalamus) and unaffected regions as internal controls

• Temporal controls: Analyze samples at different disease stages/timepoints to track ATAD3A oligomerization progression

• Genetic knockout/knockdown controls: ATAD3A heterozygous knockout models (e.g., CMV; ATAD3A fl/+) provide critical validation of antibody specificity and functional relevance

• Functional mutant controls: Include ATAD3A ATPase dead mutant (K358E) samples to distinguish between effects of oligomerization versus enzymatic activity

These controls enable robust data interpretation and help distinguish disease-specific changes from experimental artifacts.

How can I effectively use HRP-conjugated ATAD3A antibodies for subcellular localization studies?

For subcellular localization studies of ATAD3A, particularly its enrichment at mitochondria-associated membranes (MAMs) in disease models, a multi-technique approach is recommended:

• Subcellular fractionation: Isolate mitochondrial, MAM, and ER fractions using differential centrifugation protocols

• Western blot validation: Use HRP-conjugated ATAD3A antibodies alongside compartment-specific markers:

  • MAM markers: FACL4, SigmaR1

  • Mitochondrial markers: VDAC, Tom20 (outer membrane), ATPB (inner membrane), ClpP (matrix)

  • ER markers: IP3R3

• Immunofluorescence co-localization: For microscopy applications, use unconjugated ATAD3A primary antibodies with fluorescent secondary antibodies

• Proximity Ligation Assay (PLA): To detect protein-protein interactions within 10-30 nm, use ATAD3A antibodies in combination with MAM marker antibodies (e.g., FACL4)

When interpreting results, compare ATAD3A distribution patterns between normal and disease conditions to identify pathology-associated relocalization.

How can I troubleshoot high background signal when using HRP-conjugated ATAD3A antibodies?

High background signal is a common challenge with directly conjugated antibodies. To minimize this issue:

• Optimize blocking conditions: Test different blocking agents (3-5% BSA, 5% non-fat milk, commercial blockers) and extended blocking times (1-2 hours at room temperature or overnight at 4°C)

• Increase washing stringency: Extend washing steps using PBS-T or TBS-T (0.1-0.3% Tween-20) and increase the number of washes

• Titrate antibody concentration: Perform a dilution series (1:500, 1:1000, 1:2000, 1:5000) to identify optimal signal-to-noise ratio

• Reduce substrate incubation time: For Western blots, minimize ECL substrate exposure time to reduce non-specific signal

• Add protein carriers: Include 1-2% non-reactive protein (BSA) in antibody diluent to reduce non-specific binding

• Pre-absorb the antibody: Incubate with non-relevant tissue lysates to remove cross-reactive antibodies

If background persists, consider using unconjugated primary ATAD3A antibodies with separate HRP-conjugated secondary antibodies which sometimes provides better signal-to-noise ratios.

How should I interpret differences in ATAD3A oligomerization patterns between experimental conditions?

When analyzing ATAD3A oligomerization data:

• Quantitative analysis: Calculate the oligomer-to-monomer ratio rather than absolute band intensities, as this better reflects the oligomerization state

• Time-course evaluation: In Aβ-treated neuronal models, ATAD3A oligomerization increases in a time-dependent manner, with significant changes observable after 24 hours of treatment

• Regional specificity: In AD mouse models like 5XFAD, ATAD3A oligomerization shows regional specificity, with significant increases in cortex, hippocampus, and thalamus, but not in other brain regions

• Correlation with pathology: Evaluate whether ATAD3A oligomerization correlates with other AD pathological markers (Aβ deposition, tau pathology)

• Functional consequences: Link oligomerization patterns to downstream effects such as MAM hyperconnectivity, which can be assessed using proximity ligation assays between MAM components (e.g., IP3R3 and VDAC)

Significant changes in oligomerization are typically defined as a 1.5-2 fold increase in the oligomer-to-monomer ratio compared to control conditions.

How can I distinguish between specific and non-specific bands when detecting ATAD3A oligomers?

Distinguishing specific ATAD3A oligomeric bands from non-specific signals requires several validation strategies:

• Molecular weight verification: ATAD3A monomers appear at ~66 kDa and dimers at ~130 kDa; bands at unexpected molecular weights should be scrutinized

• Reducing vs. non-reducing conditions: Authentic ATAD3A oligomers should disappear or significantly decrease under reducing conditions (β-mercaptoethanol)

• Genetic validation: Samples from ATAD3A knockdown/knockout models should show reduced or absent specific bands

• Chemical cross-linking: Treatment with cross-linkers like BMH should enhance specific oligomeric bands in disease models

• Peptide competition: Pre-incubation of the antibody with immunizing peptide should eliminate specific bands

• Multiple antibodies: Validation with different ATAD3A antibodies targeting distinct epitopes

For example, in 5XFAD AD mouse models, genetic reduction of ATAD3A (5XFAD het; CMV; ATAD3A fl/+) reduces the levels of oligomeric bands to those observed in wildtype littermates, confirming their specificity .

How can HRP-conjugated ATAD3A antibodies be used to investigate MAM integrity in neurodegenerative diseases?

MAM (mitochondria-associated membranes) integrity is emerging as a critical factor in neurodegenerative pathologies. HRP-conjugated ATAD3A antibodies can be utilized in several approaches:

• Co-immunoprecipitation (co-IP): Use ATAD3A antibodies to pull down protein complexes, followed by western blotting for MAM components (requires unconjugated antibodies)

• Western blot analysis of MAM fractions: Quantify ATAD3A levels in isolated MAM fractions compared to other mitochondrial compartments

• Proximity Ligation Assay (PLA) quantification: Measure interaction between ATAD3A and MAM markers (FACL4) using PLA and quantify:

  • Number of PLA-positive puncta per cell

  • Size distribution of PLA-positive puncta

  • Intensity of PLA signals

Research has demonstrated that ATAD3A oligomerization promotes hyperconnectivity of MAMs in AD models, with both increased number and size of PLA-positive puncta between ATAD3A and FACL4 in the postmortem cortex of AD patients and 5XFAD mice . This can be reversed by ATAD3A knockdown or preventing ATAD3A oligomerization, suggesting a potential therapeutic approach.

What methodological approaches can be used to study the impact of ATAD3A oligomerization on mitochondrial function?

To investigate the functional consequences of ATAD3A oligomerization on mitochondrial biology:

• Mitochondrial morphology analysis:

  • Live-cell imaging using mitochondrial-targeted fluorescent proteins

  • Fixed-cell immunofluorescence using mitochondrial markers

  • Electron microscopy for ultrastructural analysis

• Bioenergetic assessments:

  • Oxygen consumption rate measurements using Seahorse XF analyzers

  • ATP production assays

  • Membrane potential analysis using potentiometric dyes (TMRM, JC-1)

• Calcium homeostasis:

  • Calcium imaging using genetically encoded calcium indicators targeted to mitochondria

  • Measurement of ER-mitochondria calcium transfer upon IP3R activation

• Mitochondrial quality control:

  • Analysis of mitophagy markers

  • Assessment of mitochondrial unfolded protein response

Research has shown that Aβ-induced mitochondrial fragmentation can be reduced by blocking ATAD3A oligomerization with DA1 peptide treatment , suggesting a direct link between ATAD3A oligomerization state and mitochondrial dynamics in disease models.

How can ATAD3A antibodies be used in developing potential therapeutic approaches for neurodegenerative diseases?

ATAD3A antibodies can facilitate therapeutic development in several ways:

• Target validation studies:

  • Quantify ATAD3A oligomerization in patient samples at different disease stages

  • Correlate ATAD3A oligomerization with clinical parameters and disease progression

• Compound screening:

  • Develop high-throughput ELISA-based screens for molecules that inhibit ATAD3A oligomerization

  • Use Western blotting with ATAD3A antibodies to validate hits from primary screens

• Therapeutic efficacy assessment:

  • Monitor ATAD3A oligomerization as a biomarker during treatment

  • Combine with functional readouts (MAM integrity, mitochondrial function)

• Mechanism-of-action studies:

  • Determine if therapeutic candidates (like DA1 peptide) directly bind ATAD3A

  • Evaluate if treatments normalize MAM hyperconnectivity and downstream pathologies

Recent research has demonstrated that genetic reduction of ATAD3A (heterozygous knockout) or blocking ATAD3A oligomerization with DA1 peptide reduces MAM hyperconnectivity and mitigates pathological features in AD models , highlighting ATAD3A as a promising therapeutic target.

How do different conjugated ATAD3A antibodies compare for specialized research applications?

When selecting between different conjugated ATAD3A antibodies for specialized applications:

For ATAD3A oligomerization studies in fixed tissue samples from neurodegenerative disease models, unconjugated antibodies with secondary detection systems often provide superior sensitivity, while directly conjugated antibodies offer advantages for multiplexed approaches .

What methodological considerations are important when comparing ATAD3A expression across different brain regions in AD models?

When analyzing regional differences in ATAD3A expression in AD models:

• Standardized sampling:

  • Precise anatomical identification of brain regions

  • Consistent sampling from equivalent regions across specimens

  • Serial sectioning for comprehensive analysis

• Quantification methods:

  • For immunohistochemistry: Cell counting, optical density measurements

  • For Western blot: Normalization to region-specific loading controls

  • For immunofluorescence: Mean fluorescence intensity, co-localization coefficients

• Cell type-specific analysis:

  • Co-staining with neuronal (NeuN), astrocytic (GFAP), microglial (Iba1) markers

  • Analysis of ATAD3A in different cell populations

• Data interpretation:

  • Account for regional variation in basal ATAD3A expression

  • Consider regional progression of AD pathology (Aβ deposition patterns)

  • Correlate with region-specific vulnerability

Studies have shown that ATAD3A oligomerization increases specifically in the cortex, hippocampus, and thalamus of 5XFAD AD mice, but not in other brain regions, consistent with the pattern of Aβ aggregation and human APP expression .

How can I design experiments to investigate the relationship between ATAD3A oligomerization and other AD-associated proteins?

To explore interactions between ATAD3A and other AD-relevant proteins:

• Co-localization studies:

  • Dual immunolabeling of ATAD3A with AD-associated proteins (APP, tau, presenilin)

  • Super-resolution microscopy to resolve spatial relationships

  • Quantitative co-localization analysis

• Protein-protein interaction analysis:

  • Co-immunoprecipitation of ATAD3A with AD-associated proteins

  • Proximity ligation assay (PLA) to detect in situ interactions

  • FRET/BRET approaches for live-cell interaction studies

• Functional relationship experiments:

  • Genetic manipulation of ATAD3A in AD models and assessment of effects on other AD proteins

  • Pharmacological modulation of AD proteins and measurement of impact on ATAD3A oligomerization

  • Time-course studies to establish sequence of events

Research has demonstrated that ATAD3A immunodensity is enriched in APP-immunopositive cells in the postmortem cortex of AD patients and mouse models , suggesting a potential functional relationship between these proteins that could be further explored using the approaches outlined above.