ATL30 Antibody

What are Atlastin proteins and what functions do they serve in cellular biology?

Atlastin proteins are GTPases involved in endoplasmic reticulum (ER) membrane fusion and structural maintenance. ATL2, specifically, has been shown to maintain ER homeostasis in healthy mouse mammary epithelial cells, facilitating the production and secretion of properly sized and composed lipid droplets into breast milk . Atlastins also play a critical role in macro-ER-phagy, which is essential for ER maintenance .

The ATL family includes several members with distinct tissue distribution patterns, with ATL3 being particularly well-characterized. ATL3 (atlastin GTPase 3) is a 541 amino acid protein with a calculated molecular weight of approximately 61 kDa . In normal physiological conditions, atlastin proteins help maintain the tubular ER network structure, and dysfunction has been implicated in multiple pathological conditions.

How do antibodies against ATL proteins advance scientific research?

Antibodies against ATL proteins serve as crucial tools for investigating their expression, localization, and function in various tissues and disease states. For instance, the ATL2 antibody HPA029108 has been used in immunohistochemical applications to detect ATL2-2 and potentially ATL2-3 isoforms . This has allowed researchers to correlate ATL2-2 expression with prognosis in estrogen-receptor-positive breast cancer .

For ATL3 specifically, antibodies such as 16921-1-AP have been validated for multiple applications including Western blot (WB), immunohistochemistry (IHC), immunofluorescence/immunocytochemistry (IF/ICC), immunoprecipitation (IP), and co-immunoprecipitation (CoIP) . These diverse applications enable researchers to study ATL3 from multiple analytical perspectives, enhancing our understanding of its biological roles.

What distinguishes different ATL antibodies in terms of reactivity and specificity?

Different ATL antibodies exhibit varying reactivity profiles across species and applications. For example, the ATL3 antibody (16921-1-AP) shows confirmed reactivity with human, mouse, and rat samples, with cited reactivity extending to monkey samples as well . This cross-species reactivity makes it valuable for comparative studies across model organisms.

When selecting an ATL antibody, researchers should consider:

• Target epitope (which may recognize specific isoforms)

• Host species (to avoid cross-reactivity in co-staining experiments)

• Validated applications (some antibodies perform better in certain techniques)

• Species reactivity (particularly important for translational research)

For instance, the scoring system for ATL2 antibody staining in breast tissue was based on intensity (none, weak, medium, and strong) and the estimated area of breast cells stained (<5, 5-<25, 25-<50, 50-100%) , demonstrating the importance of standardized evaluation criteria.

What are the validated applications for ATL3 antibodies in research?

ATL3 antibodies have been validated for multiple research applications, as evidenced by published literature. The 16921-1-AP ATL3 antibody has been documented in:

This antibody has been tested and found positive in Western blot analyses of multiple cell lines and tissues, including HEK-293T cells, HeLa cells, HepG2 cells, mouse liver tissue, SMMC-7721 cells, Jurkat cells, NIH/3T3 cells, and rat liver tissue . Additionally, it has shown positive results in immunoprecipitation with HeLa cells and in immunohistochemistry with human stomach cancer tissue .

What are the optimal protocols for Western blotting with ATL antibodies?

For successful Western blotting with ATL3 antibody (16921-1-AP), the recommended dilution range is 1:2000-1:12000 . The observed molecular weight of ATL3 is 61 kDa, consistent with its calculated weight . Researchers should optimize the dilution within this range based on their specific sample type and detection system.

A general protocol would include:

• Protein extraction from cells/tissues of interest

• SDS-PAGE separation (typically 10% gel works well for 61 kDa proteins)

• Transfer to PVDF or nitrocellulose membrane

• Blocking with 5% non-fat milk or BSA in TBST

• Primary antibody incubation (ATL3 antibody at 1:2000-1:12000 dilution) overnight at 4°C

• Washing with TBST (3-5 times, 5-10 minutes each)

• Secondary antibody incubation (anti-rabbit IgG) for 1-2 hours at room temperature

• Washing with TBST (3-5 times, 5-10 minutes each)

• Detection using chemiluminescence or fluorescence-based systems

The specificity of bands should be verified using positive controls from known ATL3-expressing tissues like liver or HeLa cells .

How should ATL antibodies be used in immunohistochemistry applications?

For immunohistochemistry applications with the ATL3 antibody (16921-1-AP), the recommended dilution range is 1:50-1:500 . For optimal results with ATL antibodies in IHC:

• Section preparation: Use formalin-fixed paraffin-embedded (FFPE) tissue sections of 4-6 μm thickness

• Antigen retrieval: For ATL3, it's important to use appropriate antigen retrieval methods. For instance, suggested antigen retrieval with TE buffer pH 9.0 (alternatively, citrate buffer pH 6.0 may be used)

• Blocking: Block endogenous peroxidase activity and non-specific binding

• Primary antibody incubation: Apply ATL3 antibody (1:50-1:500 dilution) and incubate for the appropriate time (typically 1 hour at room temperature or overnight at 4°C)

• Detection system: Use appropriate visualization systems (e.g., DAB for chromogenic detection)

• Counterstaining: Counterstain with hematoxylin to visualize nuclei

• Controls: Always include positive controls (tissues known to express ATL3) and negative controls (primary antibody omitted)

The optimal dilution should be determined empirically for each experimental system, as the documentation notes "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" and that dilution can be "Sample-dependent" .

What considerations are important for immunofluorescence with ATL antibodies?

For immunofluorescence/immunocytochemistry applications with ATL3 antibody, the recommended dilution range is 1:50-1:500 . The antibody has been positively validated in HeLa cells and HepG2 cells for IF/ICC applications .

Key methodological considerations for IF include:

• Sample preparation: For cultured cells, fixation with 4% paraformaldehyde preserves cellular structure while maintaining epitope accessibility

• Permeabilization: Gentle permeabilization with 0.1-0.3% Triton X-100 allows antibody access to intracellular targets

• Blocking: Use appropriate blocking solution (typically 5-10% normal serum from the same species as the secondary antibody)

• Primary antibody incubation: Apply diluted ATL3 antibody (1:50-1:500) and incubate overnight at 4°C

• Secondary antibody selection: Use fluorophore-conjugated anti-rabbit IgG that matches your microscopy setup

• Counterstaining: Nuclear counterstaining with DAPI provides context for localization

• Mounting: Use anti-fade mounting medium to prevent photobleaching

Since ATL proteins are associated with the endoplasmic reticulum, co-staining with established ER markers can help confirm proper localization and antibody specificity.

How can researchers validate the specificity of ATL antibodies?

Validating antibody specificity is critical for reliable research outcomes. For ATL antibodies, multiple approaches should be employed:

• Knockout/knockdown validation: The ATL3 antibody (16921-1-AP) has been validated in knockout/knockdown studies as mentioned in the literature references . This represents the gold standard for specificity validation.

• Immunoprecipitation followed by mass spectrometry: This can confirm that the antibody is pulling down the intended target protein.

• Pre-absorption controls: Pre-incubating the antibody with purified antigen should eliminate specific staining.

• Multiple antibodies targeting different epitopes: Consistent results with antibodies recognizing different regions of the protein increase confidence in specificity.

• Correlation with mRNA expression: Protein expression detected by the antibody should generally correlate with mRNA levels across tissues.

For ATL3 antibody specifically, the immunogen used was an ATL3 fusion protein (Ag10279) , which researchers can consider when assessing potential cross-reactivity with related proteins.

What approaches can resolve inconsistent results with ATL antibodies?

When facing inconsistent results with ATL antibodies, consider these methodological troubleshooting steps:

• Sample preparation variability: Standardize tissue fixation times, processing protocols, and antigen retrieval methods.

• Antibody titration: As recommended for the ATL3 antibody, titration is essential as optimal dilution can be sample-dependent .

• Detection system sensitivity: For weak signals, more sensitive detection systems like tyramide signal amplification may be necessary.

• Batch-to-batch variation: Record lot numbers and prepare larger aliquots of a single lot for long-term studies.

• Protein modification effects: Post-translational modifications may affect epitope accessibility; consider different lysis buffers and preparation methods.

• Storage conditions: The ATL3 antibody should be stored at -20°C with aliquoting recommended for long-term stability .

• Species-specific optimizations: While the ATL3 antibody is reactive with human, mouse, and rat samples , protocol adjustments may be necessary across species.

How should researchers approach multiplexing experiments with ATL antibodies?

For multiplexing experiments combining ATL antibodies with other markers:

• Host species selection: The ATL3 antibody (16921-1-AP) is produced in rabbit , so pair with antibodies from different host species (mouse, goat, etc.) to avoid cross-reactivity.

• Fluorophore selection: Choose fluorophores with minimal spectral overlap when designing multiplex immunofluorescence panels.

• Sequential staining: For challenging combinations, consider sequential staining with intermediate fixation steps.

• Blocking optimization: When using multiple primary antibodies, ensure adequate blocking of non-specific binding sites.

• Controls: Include single-stain controls to verify specificity and absence of bleed-through.

• Image acquisition settings: Optimize exposure settings for each channel independently.

When planning immunofluorescence co-localization studies, the subcellular localization of ATL proteins to the endoplasmic reticulum should inform the selection of complementary markers.

What strategies can address weak or non-specific signals with ATL antibodies?

To address weak or non-specific signals when working with ATL antibodies:

• Signal enhancement: For weak but specific signals:

  • Increase antibody concentration (within recommended ranges)

  • Extend incubation time

  • Use signal amplification systems

  • Optimize antigen retrieval conditions

• Reducing background: For high background/non-specific binding:

  • Improve blocking (longer times, different blocking agents)

  • Increase washing duration and stringency

  • Reduce antibody concentration

  • Use more dilute secondary antibody

• Sample-specific adjustments: As noted for the ATL3 antibody, optimal conditions can be sample-dependent , so optimization for specific tissue types may be necessary.

• Storage and handling: The ATL3 antibody is provided in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) and should be stored at -20°C . Improper storage can reduce antibody performance.

• Tissue-specific background: For IHC applications with ATL3 antibody in human stomach cancer tissue, specific antigen retrieval with TE buffer pH 9.0 is suggested , demonstrating the importance of tissue-specific optimization.

How should researchers interpret ATL protein expression patterns in normal versus diseased tissues?

When interpreting ATL protein expression patterns:

When conducting comparative studies, standardized image acquisition and analysis parameters are essential for reliable interpretation.

What controls are essential when using ATL antibodies in critical research applications?

Essential controls for ATL antibody experiments include:

• Positive controls: Use tissues or cell lines known to express the target protein. For ATL3, this would include HeLa cells, HepG2 cells, and liver tissue which have been validated .

• Negative controls: Include:

  • Primary antibody omission

  • Isotype controls (irrelevant antibodies of the same isotype and concentration)

  • Tissues known not to express the target protein

• Knockdown/knockout controls: As mentioned for the ATL3 antibody, validation in knockout/knockdown models has been published , providing a gold standard for specificity.

• Absorption controls: Pre-incubation of the antibody with immunogen (such as the ATL3 fusion protein Ag10279) should eliminate specific staining.

• Dilution series: Testing a range of antibody dilutions helps identify the optimal signal-to-noise ratio.

• Cross-validation: When possible, verify results using alternative methods (e.g., complementing IHC with Western blot).

• Internal controls: Within tissue sections, look for cell types known to be positive or negative for expression to serve as internal controls.

How can researchers integrate ATL antibody data with other molecular findings?

To integrate ATL antibody data with other molecular findings:

• Correlative analysis with transcriptomics: Compare protein expression detected by ATL antibodies with mRNA expression data to identify potential post-transcriptional regulation.

• Pathway analysis: Place ATL protein expression patterns in the context of related pathway components. For example, ATL2's role in ER homeostasis suggests examining related ER stress markers.

• Protein-protein interactions: The ATL3 antibody has been validated for co-immunoprecipitation (CoIP) , enabling studies of protein-protein interactions.

• Functional validation: Combine antibody-based detection with functional assays. For example, given ATL2's role in lipid droplet production , correlate expression with lipid metabolic phenotypes.

• Multi-omics integration: Integrate proteomics, transcriptomics, and metabolomics data to develop comprehensive models of ATL protein function.

• Structure-function analysis: Use computational antibody design tools and molecular modeling to predict how structural variations in ATL proteins might affect function .

What are the implications of ATL protein expression patterns in disease progression and therapeutic response?

The implications of ATL protein expression in disease contexts include:

• Prognostic indicators: High ATL2-2 expression has been associated with worse prognosis in estrogen-receptor-positive breast cancer , suggesting potential value as a prognostic biomarker.

• Therapeutic targets: Understanding the expression and function of ATL proteins could identify new therapeutic opportunities. For comparison, the clinical trial results for BA3011, targeting AXL, showed encouraging efficacy signals in patients who previously experienced PD-1/L1 treatment failure .

• Response prediction: Expression patterns may correlate with treatment response. In the BA3011 clinical trial example, patients with AXL TmPS of 1% still experienced a partial response , demonstrating the importance of detailed expression analysis.

• Disease mechanisms: ATL2's downregulation by miR-30e-5p in the synovial tissue of a mouse model with rheumatoid arthritis suggests potential roles in inflammatory disease processes.

• Combinatorial biomarkers: Integrating ATL expression data with other markers may provide more robust prognostic or predictive information, similar to how AXL positivity was combined with EGFR status in evaluating BA3011 efficacy .

When investigating these implications, researchers should consider longitudinal sampling to track expression changes throughout disease progression and treatment.

What emerging technologies may enhance ATL antibody applications?

Emerging technologies that could enhance ATL antibody research include:

• Computational antibody design: Advanced modeling techniques can predict antibody structure from sequence and enhance antibody-antigen interaction understanding . This could lead to more specific and higher-affinity ATL antibodies.

• Single-cell protein analysis: Technologies like Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) could allow simultaneous analysis of ATL protein expression and transcriptomics at single-cell resolution.

• Super-resolution microscopy: Techniques such as STORM or PALM can provide nanoscale resolution of ATL protein localization within the ER network.

• In situ proximity ligation: This technique could enhance detection of ATL protein interactions within their native cellular environment.

• Automated image analysis: Machine learning approaches can improve quantification and pattern recognition in ATL antibody staining, similar to scoring systems already established for ATL2 .

How might ATL antibodies contribute to personalized medicine approaches?

ATL antibodies could contribute to personalized medicine through:

• Biomarker development: Similar to how AXL expression predicted response to BA3011 therapy , ATL protein expression patterns could identify patient subgroups likely to respond to specific treatments.

• Companion diagnostics: ATL antibodies might be developed into diagnostic tests that accompany targeted therapies, especially for diseases where ER dysfunction plays a role.

• Disease subtyping: ATL expression patterns could help stratify complex diseases into molecular subtypes with distinct prognoses and treatment responses.

• Monitoring treatment efficacy: Changes in ATL protein expression or localization during treatment could provide early indicators of response or resistance.

• Target identification: Understanding ATL protein function through antibody-based studies could reveal new therapeutic targets, similar to how antibody-based studies have advanced other fields of personalized medicine.

The potential of ATL proteins as biomarkers is supported by findings that high ATL2-2 expression associates with worse prognosis in estrogen-receptor-positive breast cancer , suggesting similar applications could be developed for other ATL family members.