ATL3 Antibody

What is ATL3 and why is it important in cellular research?

ATL3 (Atlastin GTPase 3) is a member of the atlastin family of dynamin-related GTPases that functions primarily in ER membrane fusion and the maintenance of proper ER morphology. ATL3 has a canonical amino acid length of 541 residues and a molecular weight of approximately 60.5 kDa. It is predominantly localized to the endoplasmic reticulum . The protein plays critical roles in Golgi organization and protein oligomerization . Research interest in ATL3 has increased significantly since the discovery that mutations in this protein, particularly Y192C and P338R, are associated with hereditary sensory and autonomic neuropathy (HSAN) . These mutations disrupt the fusogenic capacity of ATL3, resulting in loss of ER connectivity and aberrant ER membrane tethering, which affects ER-mitochondria contact sites and mitochondrial function .

What are the key characteristics of commercially available ATL3 antibodies?

Currently available ATL3 antibodies exhibit several important characteristics that researchers should consider:

How do I select the most appropriate ATL3 antibody for my specific research application?

When selecting an ATL3 antibody, consider the following research-focused criteria:

• Experimental application: Different antibodies demonstrate varying performance across applications. For Western blotting, antibodies targeting AA 217-245 or the central region show good specificity . For immunofluorescence studies, particularly those examining ER structures, antibodies validated specifically for IF with documented subcellular localization patterns are essential .

• Target epitope relevance: Consider whether your research requires detection of specific domains or regions of ATL3. For studies of ATL3 mutations, select antibodies whose epitopes are distant from the mutation site to avoid potential detection issues .

• Species cross-reactivity: If performing comparative studies across different model organisms, choose antibodies with validated cross-reactivity. Some ATL3 antibodies react with human, mouse, and rat samples, while others have broader species reactivity .

• Validation data quality: Review the validation data provided by manufacturers, particularly the specificity in relevant cell types. For ATL3, positive WB detection has been reported in Jurkat, HEK-293, HeLa, HepG2, and SMMC-7721 cells .

What is the expected molecular weight of ATL3 in Western blot applications?

The canonical molecular weight for ATL3 in Western blot applications is approximately 60-61 kDa, consistent with its theoretical molecular weight of 60.5 kDa . In experimental validation using Western blot with ATL3 antibodies, the observed molecular weight is typically 61 kDa when detected in Jurkat cells, HEK-293 cells, HeLa cells, and HepG2 cells . When interpreting Western blot results, researchers should be aware that post-translational modifications or splice variants might result in slight deviations from this expected molecular weight. Additionally, different sample preparation methods or running conditions might affect the apparent molecular weight of the protein in SDS-PAGE.

How can ATL3 antibodies be utilized to investigate ER-mitochondria contact sites in neuropathological models?

ATL3 antibodies serve as valuable tools for investigating ER-mitochondria contact sites (MCSs) in the context of neurological disorders, particularly those associated with ATL3 mutations. Based on recent research findings:

• Proximity-based imaging approaches: Combine ATL3 antibodies with mitochondrial markers in super-resolution microscopy to quantitatively assess ER-mitochondria contacts. Studies have shown that disease-causing mutations in ATL3 (Y192C) almost double the number of MCSs between these organelles without affecting the size of individual contact sites .

• Sequential immunoprecipitation protocol: Use ATL3 antibodies in tandem with mitochondrial membrane protein antibodies to isolate and characterize the protein complexes at these contact sites. This approach can help identify changes in protein composition at MCSs in disease models.

• CLEM (Correlative Light and Electron Microscopy): Use immuno-gold labeled ATL3 antibodies for high-resolution visualization of the ultrastructure of ER-mitochondria contacts. Research has demonstrated that while the size of individual MCSs remains unchanged between wild-type and mutant ATL3 expressing cells, the fraction of mitochondrial surface in contact with the ER increases significantly in cells expressing the ATL3 Y192C mutation .

• Live-cell imaging: Combine fluorescently tagged ATL3 antibody fragments with mitochondrial tracking dyes to monitor dynamic changes in ER-mitochondria interactions, which is particularly relevant when studying the impact of ATL3 mutations on mitochondrial motility.

What are the methodological considerations when using ATL3 antibodies to study autophagy in neurodegenerative contexts?

When utilizing ATL3 antibodies to investigate the relationship between ER morphology and autophagy in neurodegenerative contexts, researchers should consider:

• Dual fluorescence assays: Implement tandem fluorescent-tagged LC3 (mCherry-GFP-LC3) assays alongside ATL3 immunostaining to distinguish between autophagosomes and autolysosomes. Research has demonstrated that cells expressing ATL3 Y192C mutation show increased co-localization of GFP and mCherry fluorescence in LC3 vesicles compared to wild-type ATL3 expressing cells, indicating altered autophagic flux .

• Temporal analysis protocol: Design time-course experiments with ATL3 antibody staining to track changes in ER morphology in relation to autophagosome formation. This is particularly important as ATL3 mutations affect ER tubule organization, which can influence autophagosome biogenesis.

• Organelle fractionation quality control: When isolating autophagic vesicles, use ATL3 antibodies to assess potential ER membrane contamination, which is crucial for accurate interpretation of biochemical analyses of autophagy-related proteins.

• Starvation-induced autophagy assessment: Compare baseline and starvation-induced autophagy between wild-type and mutant ATL3 expressing cells using appropriate antibodies. Studies have shown differential responses to starvation conditions between cells expressing wild-type ATL3 and those with the Y192C mutation .

• Co-immunoprecipitation strategy: Employ ATL3 antibodies in co-IP experiments to identify potential interactions with autophagy-related proteins, which may reveal novel mechanisms linking ER morphology defects to autophagy dysregulation.

How can I effectively use ATL3 antibodies to distinguish between normal and pathological ER membrane phenotypes?

To effectively distinguish between normal and pathological ER membrane phenotypes using ATL3 antibodies:

• High-resolution phenotypic analysis: Employ super-resolution microscopy techniques (such as STED, SIM, or STORM) combined with ATL3 immunostaining to visualize subtle changes in ER tubule organization. Research has shown that HSAN-causing mutations in ATL3 result in tangling of ER tubules due to aberrant membrane tethering, which can be detected using high-resolution imaging .

• Quantitative morphometric protocol: Develop automated image analysis workflows that quantify parameters such as:

  • ER tubule length and branching frequency

  • Tubule diameter consistency

  • Junction distribution patterns

  • Three-way junction abundance

  These measurements can provide objective metrics for distinguishing normal from pathological ER networks.

• Co-localization with ER domain markers: Use dual immunostaining with ATL3 antibodies and markers for different ER domains (rough ER, smooth ER, ER exit sites) to assess domain-specific alterations in ER organization. This approach can reveal whether ATL3 mutations affect specific ER subdomains preferentially.

• Dynamic ER remodeling assessment: Combine fixed-cell ATL3 immunostaining with live-cell imaging of ER dynamics to correlate structural phenotypes with functional defects in ER remodeling and fusion events.

What are the optimal fixation and permeabilization protocols for ATL3 immunofluorescence in different cell types?

The optimal conditions for ATL3 immunofluorescence vary by cell type and specific research question:

For optimal results with ATL3 immunofluorescence:

• Fixation timing is critical: Over-fixation can mask epitopes. For PFA fixation, strictly adhere to the recommended time (typically 10-15 minutes).

• Buffer composition matters: Use PBS with calcium and magnesium for fixation to better preserve ER structure when using ATL3 antibodies.

• Sequential antibody application: When co-staining with other ER markers, apply the ATL3 antibody first (1:10-1:100 dilution) followed by other antibodies to ensure optimal epitope accessibility .

• Validation controls: Always include cells with known ATL3 expression patterns (e.g., HepG2 cells) as positive controls when optimizing protocols for new cell types .

What are the recommended protocols for optimizing ATL3 antibody-based Western blot assays?

For optimal Western blot results with ATL3 antibodies, consider the following protocol recommendations:

• Sample preparation optimization:

  • Use RIPA buffer supplemented with protease inhibitors for cell lysis

  • For tissue samples, particularly mouse liver tissue which has been validated with ATL3 antibodies, homogenize in buffer containing 250 mM sucrose, 20 mM HEPES-KOH (pH 7.4), and protease inhibitors

  • Heat samples at 70°C (not 95°C) for 10 minutes to prevent aggregation of this membrane protein

• Gel selection and transfer parameters:

  • Use 8-10% acrylamide gels for optimal resolution of the 61 kDa ATL3 protein

  • Transfer to PVDF membranes using semi-dry transfer (15V, 30 minutes) or wet transfer (30V overnight at 4°C)

  • Validate transfer efficiency with reversible protein staining before blocking

• Antibody incubation protocol:

  • Block membranes in 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Dilute primary ATL3 antibody 1:500-1:5000 in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

  • Wash 4x10 minutes with TBST

  • Incubate with HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

• Detection optimization:

  • Use enhanced chemiluminescence detection with exposure times starting at 30 seconds

  • For lower abundance detection, consider using signal enhancement systems or fluorescent secondary antibodies with digital imaging

• Validated controls:

  • Positive controls: Jurkat cells, HEK-293 cells, HeLa cells, and HepG2 cells have all demonstrated reliable ATL3 detection

  • Loading control: Select appropriate loading controls based on experimental context (β-actin, GAPDH, or tubulin)

What strategies can be employed for validating ATL3 antibody specificity in experimental systems?

Comprehensive validation of ATL3 antibody specificity requires multiple complementary approaches:

• Genetic validation approaches:

  • CRISPR/Cas9 knockout of ATL3: Generate ATL3-null cell lines to confirm absence of signal with the antibody

  • siRNA/shRNA knockdown: Perform partial knockdown to demonstrate proportional reduction in signal intensity

  • Overexpression validation: Express tagged versions of ATL3 and confirm co-localization with antibody signal

• Peptide competition assay protocol:

  • Pre-incubate the ATL3 antibody with excess immunizing peptide (when available)

  • In parallel, use the antibody without peptide competition

  • Compare signals - specific binding should be abolished or significantly reduced in the peptide-competed sample

• Multi-antibody concordance testing:

  • Use multiple antibodies targeting different epitopes of ATL3 (e.g., antibodies targeting AA 1-187, AA 217-245, and AA 360-440)

  • Compare signal patterns across different applications (WB, IF, IP)

  • Consistent results across different antibodies increase confidence in specificity

• Cross-species validation strategy:

  • Test the antibody in samples from multiple species where cross-reactivity is claimed

  • Differences in non-conserved regions should result in predictable species-specific patterns

  • Many ATL3 antibodies have validated reactivity with human, mouse, and rat samples

• Mass spectrometry verification:

  • Perform immunoprecipitation using the ATL3 antibody

  • Analyze the precipitated proteins by mass spectrometry

  • Confirm the presence of ATL3 peptides and assess the presence of off-target proteins

How can I address non-specific binding issues with ATL3 antibodies in immunofluorescence applications?

When encountering non-specific binding with ATL3 antibodies in immunofluorescence applications, consider the following troubleshooting approaches:

• Optimized blocking protocol:

  • Test different blocking agents (BSA, normal serum, commercial blocking buffers)

  • Extend blocking time to 2 hours at room temperature

  • Add 0.1-0.3% Triton X-100 to the blocking solution to reduce hydrophobic interactions

  • Consider dual blocking with 5% BSA followed by 5% normal serum from the secondary antibody species

• Antibody dilution optimization:

  • Perform a dilution series beyond the recommended range (1:10-1:100)

  • Extend primary antibody incubation to overnight at 4°C with more dilute solutions

  • Prepare antibody dilutions in blocking buffer containing 0.05% Tween-20

• Modified washing protocol:

  • Increase wash stringency with higher salt PBS (up to 500 mM NaCl)

  • Extend wash steps to 5 x 10 minutes with gentle agitation

  • Add 0.1% Tween-20 to wash buffers to reduce non-specific hydrophobic interactions

• Peptide pre-absorption strategy:

  • If the immunizing peptide is available, pre-absorb the antibody with excess peptide

  • Include a gradient of peptide concentrations to identify optimal conditions

  • Compare staining patterns to identify which signals are eliminated (specific) versus retained (non-specific)

• Fluorophore considerations:

  • Switch to a different fluorophore if autofluorescence is an issue in your sample

  • Consider directly conjugated primary antibodies to eliminate secondary antibody cross-reactivity

  • Use Sudan Black B treatment (0.1% in 70% ethanol) after secondary antibody incubation to reduce background fluorescence

What are the potential causes and solutions for weak or absent ATL3 signal in Western blot applications?

When troubleshooting weak or absent ATL3 signals in Western blot applications:

When optimizing for ATL3 detection, consider that membrane proteins like ATL3 can be challenging to extract and may require specialized extraction buffers containing mild detergents like 1% digitonin or 0.5% DDM (n-dodecyl-β-D-maltoside).

How can I optimize immunoprecipitation protocols for ATL3 to study its protein interaction partners?

For successful immunoprecipitation of ATL3 and identification of its interaction partners:

• Optimized cell lysis protocol:

  • Use mild lysis buffers to preserve protein-protein interactions

  • Recommended buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40 substitute, 0.5% sodium deoxycholate with protease inhibitors

  • Perform lysis on ice for 30 minutes with gentle agitation

  • Clear lysates by centrifugation at 14,000 × g for 15 minutes at 4°C

• Antibody selection and binding strategy:

  • Choose antibodies validated for IP applications (such as those diluted 1:500-1:5000 for IP)

  • For co-IP of membrane protein complexes, consider membrane-specific extraction methods

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Use 2-5 μg of antibody per 500 μg-1 mg of total protein

  • Incubate antibody with lysate for 4 hours to overnight at 4°C with gentle rotation

• Bead selection and washing optimization:

  • Compare protein A, protein G, or combination A/G beads based on antibody isotype

  • For stringent washing: 4 washes with lysis buffer followed by 2 washes with higher salt buffer (300 mM NaCl)

  • For preserving weaker interactions: 6 gentle washes with lysis buffer only

  • Consider crosslinking the antibody to beads to avoid antibody contamination in eluted samples

• Elution strategies:

  • Standard elution: Boil in 1× Laemmli buffer at 70°C for 10 minutes

  • Native elution: Use excessive immunizing peptide to compete for antibody binding

  • Acidic elution: 0.1 M glycine pH 2.5, immediately neutralized with 1 M Tris pH 8.0

  • For mass spectrometry analysis: Consider on-bead digestion to reduce contaminants

• Validation of results:

  • Include IgG control IP processed identically to the ATL3 IP

  • Perform reciprocal IPs with antibodies against suspected interaction partners

  • Consider proximity labeling approaches (BioID, APEX) as complementary techniques

Successful ATL3 IP has been documented in HeLa cells using specific antibodies, with the observed molecular weight of 61 kDa in subsequent Western blot analysis .

How are ATL3 antibodies being utilized to investigate the molecular mechanisms of hereditary sensory neuropathy?

ATL3 antibodies are enabling several research strategies to elucidate the pathophysiology of hereditary sensory neuropathy:

• Comparative proteomics approaches: ATL3 antibodies are being used to immunoprecipitate wild-type and mutant (Y192C, P338R) ATL3 protein complexes from patient-derived cells, followed by mass spectrometry analysis to identify differential protein interactions that may contribute to disease pathology .

• Structure-function studies: By comparing immunolocalization patterns of wild-type versus mutant ATL3 in patient fibroblasts, researchers have demonstrated that disease-causing mutations result in altered ER morphology and distribution, specifically causing aberrant ER membrane tethering and tangling of ER tubules .

• ER-mitochondria contact site quantification: Using ATL3 antibodies alongside mitochondrial markers, researchers have shown that HSAN-causing mutations increase the number of ER-mitochondria contact sites, which affects mitochondrial function and motility .

• Autophagy flux assessment: ATL3 antibodies combined with autophagy markers have revealed that cells expressing mutant ATL3 (Y192C) display altered autophagic processing, with increased co-localization of GFP and mCherry in tandem fluorescent-tagged LC3 assays, indicating changes in autophagic flux that may contribute to neuronal pathology .

• Neuronal subcellular dynamics: In neuronal models, ATL3 antibodies are helping to map the distribution and dynamics of mutant ATL3 in axons and dendrites, providing insights into why sensory neurons are particularly vulnerable to ATL3 mutations.

What are the emerging applications of ATL3 antibodies in neurodegenerative disease research beyond hereditary sensory neuropathy?

ATL3 antibodies are finding broader applications in neurodegenerative disease research:

• ER stress pathway investigation: By co-immunostaining for ATL3 and ER stress markers (BiP/GRP78, CHOP, XBP1), researchers are exploring whether alterations in ER morphology mediated by ATL3 dysfunction contribute to ER stress in various neurodegenerative conditions.

• Mitochondrial dysfunction models: The finding that ATL3 mutations affect ER-mitochondria contact sites has prompted investigation of ATL3's role in other neurodegenerative diseases where mitochondrial dysfunction is implicated, such as Parkinson's and Alzheimer's diseases.

• ER-phagy regulation studies: ATL3 antibodies are being used to investigate the role of proper ER morphology in selective ER autophagy (ER-phagy), which is increasingly recognized as important in neurodegenerative diseases.

• Axonal transport mechanisms: By studying the distribution and dynamics of ATL3 in neuronal processes, researchers are gaining insights into how ER structure affects axonal transport, a process commonly disrupted in multiple neurodegenerative conditions.

• Therapeutic target validation: ATL3 antibodies are enabling the validation of ATL3 and its interacting partners as potential therapeutic targets, allowing researchers to assess the effects of experimental compounds on ATL3 localization, ER morphology, and downstream cellular functions.

These emerging applications highlight the growing importance of ATL3 antibodies as tools for understanding fundamental mechanisms of ER biology that may be relevant across multiple neurodegenerative disease contexts.