ATAD3A Antibody

What is ATAD3A and why is it important in cellular function?

ATAD3A is a mitochondrial membrane-bound ATPase that plays essential roles in mitochondrial network organization, metabolism, and cellular growth. It is particularly important for embryonic development, with de novo mutations causing neurological syndromes characterized by developmental delay . The protein forms part of the mitochondrial machinery that maintains proper organelle function and dynamics. ATAD3A is notably enriched in mitochondrial-associated membranes (MAMs), suggesting its involvement in ER-mitochondria communication .

What are the key characteristics of ATAD3A protein?

ATAD3A is a 634 amino acid protein with a molecular weight of approximately 71.4 kDa (though often observed at around 68 kDa in experimental conditions) . It belongs to the AAA ATPase protein family and localizes primarily to the mitochondria. The protein has three known isoforms resulting from alternative splicing . ATAD3A shows notable expression in lung adenocarcinomas and is also referred to by synonyms including PHRINL and ATPase family AAA domain-containing protein 3A .

How does ATAD3A differ from ATAD3B?

ATAD3A and ATAD3B are related proteins that share significant sequence homology, requiring careful antibody selection when targeting either protein specifically. Some antibodies (such as 16610-1-AP) target both ATAD3A and ATAD3B . While both proteins are localized to mitochondria, they may serve complementary but distinct functions in mitochondrial dynamics and maintenance. Research often requires distinguishing between these proteins to understand their specific roles.

What applications are ATAD3A antibodies commonly used for?

ATAD3A antibodies are employed in multiple applications including Western Blot (WB), immunohistochemistry (IHC), immunofluorescence (IF), immunoprecipitation (IP), co-immunoprecipitation (CoIP), and ELISA . These diverse applications allow researchers to study ATAD3A expression levels, localization patterns, protein-protein interactions, and potential alterations in disease states. Most commercially available antibodies show reactivity with human, mouse, and rat samples .

What are the recommended dilutions for different applications of ATAD3A antibodies?

Recommended dilutions for ATAD3A antibodies vary by application. Based on the search results, typical dilutions include:

It's important to note that optimal dilutions may be sample-dependent and should be determined empirically for each experimental system .

How can I validate the specificity of ATAD3A antibodies?

Validating ATAD3A antibody specificity requires multiple approaches. First, compare observed molecular weight (approximately 68-72 kDa) with the expected size. Second, include positive controls from cells known to express ATAD3A (such as MAF or NCCIT cells) . Third, employ knockout/knockdown validation - several publications have used ATAD3A knockdown to confirm antibody specificity . Finally, conduct peptide competition assays using the immunogen peptide. Cross-reactivity with ATAD3B should be considered when interpreting results.

What sample preparation methods are optimal for detecting ATAD3A in different applications?

For Western blot applications, standard protein extraction methods with mitochondrial enrichment protocols can enhance ATAD3A detection. For immunohistochemistry, antigen retrieval with TE buffer pH 9.0 is suggested, although citrate buffer pH 6.0 may serve as an alternative . For immunofluorescence studies, paraformaldehyde fixation followed by permeabilization with Triton X-100 is generally effective. When studying ATAD3A oligomerization, non-reducing conditions (absence of β-mercaptoethanol) are necessary to preserve oligomeric structures .

How can I visualize ATAD3A localization in mitochondria using immunofluorescence?

For optimal visualization of ATAD3A in mitochondria using immunofluorescence, co-stain with established mitochondrial markers (such as VDAC, Tom20, or MitoTracker dyes) to confirm mitochondrial localization. Use confocal microscopy for high-resolution imaging of mitochondrial networks. When studying ATAD3A specifically at MAMs, proximity ligation assays (PLA) between ATAD3A and MAM markers (like SigmaR1 or IP3R3) can be performed, as demonstrated in AD research . Counterstaining with neuronal markers (such as NeuN) can help evaluate ATAD3A expression specifically in neurons within brain tissues .

How does ATAD3A oligomerization contribute to neurodegenerative pathology?

ATAD3A forms oligomers (primarily dimers) under pathological conditions, exhibiting a gain-of-function mechanism that promotes neuropathology. In Alzheimer's disease models, ATAD3A oligomerization significantly increases at the mitochondria-associated membranes (MAMs) . This oligomerization enhances ER-mitochondria tethering and MAM hyperconnectivity, which are implicated in AD pathogenesis. Notably, reducing ATAD3A levels through heterozygous knockout or blocking ATAD3A oligomerization with specific peptides (such as DA1) reduces these pathological changes in AD models, suggesting a direct mechanistic link between ATAD3A oligomerization and neurodegeneration .

What methods can detect ATAD3A oligomerization in disease models?

To detect ATAD3A oligomerization, researchers can employ several approaches: (1) Western blotting under non-reducing conditions (without β-mercaptoethanol), which preserves disulfide bonds critical for ATAD3A dimerization; (2) Chemical cross-linking with agents like bismaleimidohexane (BMH) followed by Western blotting; (3) Mitochondrial subcompartmental fractionation to assess ATAD3A enrichment in MAM fractions; and (4) Proximity ligation assays (PLA) using antibodies against ATAD3A and other MAM proteins to visualize protein-protein interactions at the ER-mitochondria interface . These techniques have successfully demonstrated increased ATAD3A oligomerization in various AD models including primary neurons treated with oligomeric Aβ, APP mutant-expressing cell lines, 5XFAD mouse brains, and postmortem brain tissue from AD patients .

How does ATAD3A contribute to mitochondrial morphology and function?

ATAD3A plays a critical role in regulating mitochondrial morphology and function. Patient fibroblasts harboring ATAD3A mutations display elongated, unbranched mitochondria and mitochondrial hyperpolarization, characteristic of hyperfused mitochondria . ATAD3A alterations affect the balance of mitochondrial dynamics proteins, with reduced levels of the fission protein Drp1 observed in patient cells . In neurons derived from patient iPSCs, ATAD3A mutations cause altered mitochondrial networks with more uniform mitochondrial staining in neuronal extensions and perinuclear accumulations . Additionally, ATAD3A oligomerization in AD models promotes mitochondrial fragmentation, which can be rescued by blocking ATAD3A oligomerization with the DA1 peptide .

What is the relationship between ATAD3A and other mitochondrial proteins in disease contexts?

ATAD3A interactions with other mitochondrial proteins are remodeled in disease contexts. In Alzheimer's disease, ATAD3A oligomerization correlates with altered levels of MAM-associated proteins, including increased IP3R3 and FACL4 . ATAD3A knockdown prevents these changes, suggesting ATAD3A acts upstream of other MAM proteins in disease pathways. In cells with ATAD3A mutations, the ratio of respiratory complex IV and II subunits is altered, alongside reduced levels of the mitochondrial fission protein Drp1 . ATAD3A localizes to MAMs along with VDAC and SigmaR1, and the distribution of ATAD3A to MAMs increases significantly in AD models . These findings suggest ATAD3A serves as a critical node in mitochondrial protein networks that become dysregulated in neurodegenerative diseases.

How should researchers approach experimental design when studying ATAD3A in disease models?

When designing experiments to study ATAD3A in disease models, researchers should implement a multi-level approach. First, establish appropriate disease models that recapitulate ATAD3A pathology - options include patient-derived fibroblasts, iPSC-derived neurons, transgenic mice (such as 5XFAD for AD studies), and cell lines treated with disease-relevant stressors (e.g., oligomeric Aβ) . Second, employ complementary techniques to assess ATAD3A oligomerization, including non-reducing Western blots and chemical cross-linking . Third, include region-specific analyses, as ATAD3A changes may be localized to specific brain regions (e.g., cortex, hippocampus, and thalamus in AD models) . Finally, incorporate both gain-of-function (overexpression) and loss-of-function (knockdown/knockout) approaches to establish causality between ATAD3A alterations and disease phenotypes.

What controls are essential when performing ATAD3A antibody-based experiments?

Essential controls for ATAD3A antibody experiments include: (1) Positive controls using cell lines known to express ATAD3A, such as MAF or NCCIT cells ; (2) Negative controls through ATAD3A knockdown/knockout validation to confirm antibody specificity; (3) Loading controls specific to the subcellular compartment being studied (e.g., VDAC or Tom20 for mitochondrial outer membrane) ; (4) For oligomerization studies, include both reducing and non-reducing conditions in parallel to demonstrate specificity of oligomeric bands; (5) When studying ATAD3A in specific cell types (e.g., neurons), include co-staining with cell-type specific markers ; and (6) When examining MAM localization, include proper subcellular fractionation controls and PLA negative controls.

How can researchers distinguish between ATAD3A expression changes and oligomerization effects?

Distinguishing between ATAD3A expression changes and oligomerization effects requires parallel assessment of multiple parameters. First, measure both mRNA and total protein levels to determine if changes are transcriptional or post-translational. In 5XFAD AD mouse models, while ATAD3A immunodensity increased, mRNA and total protein levels remained comparable to controls, suggesting post-translational modifications like oligomerization rather than expression changes . Second, compare ATAD3A levels under reducing versus non-reducing conditions - increased ATAD3A signal under non-reducing conditions without changes in monomer levels under reducing conditions indicates oligomerization without expression changes. Third, use chemical cross-linkers like BMH to stabilize and detect protein-protein interactions. Finally, assess ATAD3A ATPase activity independently, as oligomerization may occur without changes in enzymatic function .

What are promising therapeutic approaches targeting ATAD3A in neurodegenerative diseases?

Emerging therapeutic approaches targeting ATAD3A in neurodegenerative diseases focus on modulating its oligomerization. The DA1 peptide, specifically designed to block ATAD3A oligomerization, has shown promise in reducing pathological MAM hyperconnectivity and mitochondrial fragmentation in AD models . Genetic approaches, such as heterozygous knockout of ATAD3A, have successfully reduced ATAD3A oligomerization in 5XFAD mice to levels comparable with wild-type littermates . Future therapeutic development may include small molecule inhibitors of ATAD3A oligomerization, targeted reduction of ATAD3A at MAMs, or restoration of proper ER-mitochondria tethering downstream of ATAD3A. Given ATAD3A's essential functions, therapies will need to normalize pathological ATAD3A activity without completely eliminating its physiological roles.

How might new antibody technologies advance ATAD3A research?

Advanced antibody technologies could significantly enhance ATAD3A research through several innovations. First, development of conformation-specific antibodies that selectively recognize oligomeric versus monomeric ATAD3A would enable direct assessment of oligomerization states without relying on non-reducing conditions. Second, antibodies with improved specificity to distinguish between ATAD3A and ATAD3B would resolve current cross-reactivity issues. Third, antibodies conjugated to proximity-based enzymes could facilitate in situ detection of ATAD3A interactions with other MAM proteins. Fourth, super-resolution microscopy-compatible antibodies would enable nanoscale visualization of ATAD3A localization within mitochondrial subcompartments. Finally, development of intrabodies (intracellular antibodies) against ATAD3A could enable real-time monitoring of ATAD3A dynamics and targeted disruption of specific ATAD3A interactions in living cells.