ATL1 Antibody

What is ATL1 and why is it important in neurological research?

ATL1 encodes atlastin-1, a critical GTPase that plays an essential role in maintaining the structure and function of the endoplasmic reticulum. This protein has garnered significant research interest because mutations in ATL1 are one of the most common causes of hereditary spastic paraplegia (HSP) . Atlastin-1's function is regulated through various post-translational modifications, including ubiquitination. Research has shown that the E3 ubiquitin ligase SYVN1 can ubiquitinate atlastin-1, thereby inhibiting its GTPase activity and affecting endoplasmic reticulum morphology . Understanding ATL1's role through antibody-based detection methods is therefore crucial for advancing our knowledge of HSP pathogenesis and potential therapeutic approaches.

What are the common applications for ATL1 antibodies in laboratory research?

ATL1 antibodies are versatile tools employed in multiple experimental techniques. The most common applications include Western Blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), Immunohistochemistry (IHC), and Immunofluorescence (IF) . Different ATL1 antibodies may show varying performance across these applications, with some optimized for specific techniques. For example, the rabbit polyclonal ATL1 antibody (31924-1-AP) has been validated for Western Blot and ELISA applications, demonstrating reactivity with human, rat, and pig samples . Researchers should select appropriate antibodies based on their specific experimental needs and the species being studied.

How do different ATL1 antibodies vary in their epitope recognition, and how does this impact experimental design?

ATL1 antibodies are designed to target various regions (epitopes) of the atlastin-1 protein, which can significantly impact their performance in different applications. Available antibodies include those targeting the N-terminal region (AA 1-100), mid-regions (AA 220-350), and C-terminal domains (AA 477-504) . When designing experiments, researchers must consider which protein domain is most accessible in their experimental conditions. For instance, some epitopes may be masked in fixed tissues but exposed in denatured protein samples for Western Blot. The choice between monoclonal antibodies (like clone 1F6B12) versus polyclonal antibodies should be guided by whether specificity or broad epitope recognition is more important for the research question . Additionally, post-translational modifications such as ubiquitination might affect epitope accessibility, particularly in studies investigating ATL1 regulation by the E3 ubiquitin ligase SYVN1 .

What are the optimal antigen retrieval methods for ATL1 detection in fixed tissues?

• Enzymatic antigen retrieval using Proteinase K solution for 10 minutes at room temperature

• HIER with retrieval buffer pH 9.0 using similar pressure boiler conditions

These alternative methods should be considered when standard protocols yield suboptimal staining or high background . The choice between these methods depends on the specific tissue type, fixation conditions, and the particular ATL1 antibody being used. Comparative optimization is recommended for critical experiments, especially when studying tissues with potential ATL1 mutations related to hereditary spastic paraplegia.

How can researchers distinguish between specific and non-specific binding when using ATL1 antibodies?

Distinguishing specific from non-specific binding is critical for accurate data interpretation when using ATL1 antibodies. Researchers should implement multiple validation strategies:

• Use appropriate negative controls, including isotype controls that match the host species and antibody class of the ATL1 antibody being used

• Perform peptide competition assays where the antibody is pre-incubated with the immunizing peptide to block specific binding

• Compare staining patterns across multiple antibodies targeting different ATL1 epitopes

• Include tissue samples known to be negative or positive for ATL1 expression

• For critical findings, validate with orthogonal techniques (e.g., mass spectrometry or mRNA analysis)

The observed molecular weight discrepancy between calculated (64 kDa) and actual detection (50 kDa) should be considered when evaluating Western Blot results . Additionally, cross-reactivity testing using samples from different species can provide valuable information about antibody specificity, as demonstrated with the documented cross-reactivity of certain ATL1 antibodies with human, rat, and pig samples .

What is the recommended immunohistochemistry protocol for ATL1 antibodies?

The optimal immunohistochemistry protocol for ATL1 antibodies involves several critical steps:

• Deparaffinization: Paraffin sections (4 μm thickness) should be baked overnight at 50°C, followed by deparaffinization and hydration in xylene and graded ethanol. During hydration, block endogenous peroxidase activity with 0.3% H₂O₂ in 95% ethanol for 5 minutes .

• Antigen Retrieval: Perform Heat Induced Epitope Retrieval (HIER) using retrieval buffer pH 6.1 in a pressure boiler at 110°C for 20 minutes. Allow slides to cool to 90°C in the pressure boiler with a total processing time of approximately 60 minutes .

• Immunostaining Program:

  • Rinse in wash buffer

  • Block with Ultra V Block for 5 minutes

  • Incubate with primary ATL1 antibody for 30 minutes at room temperature

  • For monoclonal antibodies, include a Primary Antibody Enhancer step for 20 minutes

  • Incubate with labeled polymer for 30 minutes

  • Develop with DAB solution for 5 minutes

  • Counterstain with hematoxylin for 5 minutes

Optimization of primary antibody dilution is essential, with recommended starting dilutions of 1:1000-1:4000 for Western Blot applications . For IHC, the optimal dilution must be determined empirically by each laboratory based on their specific conditions and detection system.

What are the appropriate positive and negative controls for ATL1 antibody validation?

Proper experimental controls are essential for validating ATL1 antibody specificity and sensitivity:

Positive Controls:

• Rat brain tissue, fetal human brain tissue, and pig brain tissue have been validated as positive controls for ATL1 expression

• These tissues demonstrate reliable ATL1 detection with antibodies like the rabbit polyclonal 31924-1-AP

Negative Controls:

• Tissues known not to express ATL1 based on transcriptomic data

• Primary antibody omission controls to assess non-specific binding of secondary detection reagents

• Isotype controls using non-specific antibodies of the same isotype (e.g., IgG1 for monoclonal 1F6B12)

For additional validation, researchers can consider using ATL1 knockdown or knockout samples where available, or peptide competition assays to confirm binding specificity.

How should researchers optimize Western Blot conditions for ATL1 detection?

Optimizing Western Blot conditions for ATL1 detection requires attention to several key parameters:

• Sample Preparation: Given ATL1's role as a GTPase associated with the endoplasmic reticulum, proper cell lysis buffers containing appropriate detergents are essential for complete protein extraction. For brain tissue samples (where ATL1 is highly expressed), specialized neural tissue lysis protocols may be required.

• Antibody Selection and Dilution: For Western Blot applications, antibodies like the rabbit polyclonal 31924-1-AP have been validated at dilutions ranging from 1:1000 to 1:4000 . Researchers should perform preliminary dilution series to determine optimal concentration for their specific sample type.

• Molecular Weight Considerations: While the calculated molecular weight of ATL1 is 64 kDa, the observed molecular weight is typically around 50 kDa . Researchers should be aware of this discrepancy when identifying ATL1 bands.

• Transfer Conditions: Due to the size of ATL1, standard semi-dry or wet transfer protocols are appropriate, with recommended transfer times of 60-90 minutes at 100V for wet transfer systems.

• Blocking Conditions: A 5% non-fat dry milk or 3-5% BSA in TBST is typically effective for reducing background when detecting ATL1.

The specific reactivity with human, rat, and pig samples should be considered when selecting positive controls , with rat brain tissue being particularly reliable for ATL1 detection.

How can ATL1 antibodies contribute to hereditary spastic paraplegia (HSP) research?

ATL1 antibodies are instrumental in HSP research because mutations in ATL1 are one of the most common causes of this neurodegenerative disorder . These antibodies enable several critical research applications:

• Mutation Impact Assessment: ATL1 antibodies can be used to evaluate how different mutations affect protein expression, localization, and post-translational modifications. This is particularly valuable for comparing wild-type and mutant ATL1 in patient-derived samples or model systems.

• Interaction Studies: Using co-immunoprecipitation with ATL1 antibodies, researchers can investigate how HSP-associated mutations alter ATL1's interaction with other proteins, particularly those involved in endoplasmic reticulum morphology and function.

• Therapeutic Development Monitoring: In pre-clinical studies of potential HSP therapies, ATL1 antibodies provide essential tools for monitoring changes in protein expression, localization, or modification in response to treatment.

• Biomarker Development: ATL1 detection in accessible patient samples might serve as a biomarker for disease progression or treatment response in some forms of HSP.

When studying ATL1 in the context of HSP, researchers should consider antibodies targeting specific domains where disease-causing mutations cluster, as these regions may show altered accessibility or expression in pathological states.

What considerations are important when using ATL1 antibodies to study the ubiquitination regulation pathway?

When investigating ATL1 regulation by ubiquitination, particularly involving the E3 ubiquitin ligase SYVN1 , researchers should consider several methodological aspects:

• Epitope Accessibility: Ubiquitination may mask certain epitopes, so antibodies targeting different regions of ATL1 should be tested to ensure detection of both modified and unmodified forms.

• Experimental Conditions: Proteasome inhibitors (e.g., MG132) may be necessary to stabilize ubiquitinated ATL1 species prior to cell lysis and immunoprecipitation.

• Detection Strategies: For Western Blot analysis of ubiquitinated ATL1, researchers should look for higher molecular weight bands (beyond the standard 50 kDa observed size) representing ubiquitin-conjugated forms.

• Antibody Combinations: Co-immunostaining or sequential probing with antibodies against ATL1 and ubiquitin can help confirm ubiquitinated species.

• Controls: Experiments manipulating SYVN1 expression (overexpression or knockdown) should be included to demonstrate the specific relationship between this E3 ligase and ATL1 ubiquitination.

The interplay between ATL1's GTPase activity and its regulation by ubiquitination provides important insights into the molecular mechanisms underlying endoplasmic reticulum morphology and potentially the pathogenesis of hereditary spastic paraplegia.

What are common challenges when using ATL1 antibodies in immunohistochemistry, and how can they be addressed?

Researchers frequently encounter several challenges when using ATL1 antibodies for immunohistochemistry:

• High Background Staining: This may result from insufficient blocking or non-specific antibody binding. Solutions include:

  • Extending the blocking step with Ultra V Block beyond the standard 5 minutes

  • Increasing wash steps duration and number

  • Further diluting the primary antibody

  • Using a more specific detection system

• Weak or Absent Staining: This may indicate issues with epitope accessibility or antibody sensitivity. Approaches include:

  • Testing alternative antigen retrieval methods, such as switching between pH 6.1 buffer and pH 9.0 buffer

  • Trying enzymatic retrieval with Proteinase K for 10 minutes at room temperature

  • Extending primary antibody incubation time beyond 30 minutes

  • Using signal amplification systems, particularly for monoclonal antibodies where Primary Antibody Enhancer can be beneficial

• Inconsistent Staining: This may reflect tissue fixation variability. Standardize fixation protocols and consider:

  • Adjusting fixation time for future samples

  • Modifying antigen retrieval time and temperature

  • Testing multiple ATL1 antibodies targeting different epitopes

• Non-specific Binding: Validate with proper controls and consider pre-absorption of antibodies with non-specific blocking proteins.

For all troubleshooting approaches, comparing results with validated positive control tissues (rat brain, fetal human brain, or pig brain tissue) provides essential reference points.

How can researchers distinguish between true ATL1 signal and artifacts in Western Blot analysis?

Distinguishing genuine ATL1 signal from artifacts in Western Blot requires systematic validation:

• Molecular Weight Verification: While the calculated molecular weight of ATL1 is 64 kDa, the experimentally observed weight is typically 50 kDa . Bands at significantly different sizes should be scrutinized as potential artifacts.

• Multiple Antibody Confirmation: Use antibodies targeting different ATL1 epitopes to confirm that the same band is recognized.

• Positive Control Comparison: Include validated positive controls such as rat brain tissue, which has been shown to express ATL1 detectable by antibodies like 31924-1-AP .

• Specificity Controls:

  • Peptide competition assays where pre-incubation of the antibody with immunizing peptide should eliminate specific bands

  • Loading gradient to demonstrate signal intensity corresponding to protein amount

• Knockout/Knockdown Validation: Where possible, include samples with genetic ablation or reduction of ATL1 expression to confirm band identity.

• Protein Extraction Method Assessment: Compare different extraction protocols as ATL1's association with the endoplasmic reticulum may require specific detergent conditions for optimal solubilization.

For optimal Western Blot performance, the recommended dilution range of 1:1000-1:4000 for antibodies like 31924-1-AP provides a starting point, though this should be empirically optimized for each experimental system .

How can ATL1 antibodies be adapted for multi-parameter analysis with other neurological markers?

ATL1 antibodies can be effectively incorporated into multi-parameter analyses alongside other neurological markers through several advanced techniques:

• Multiplex Immunofluorescence: By selecting ATL1 antibodies from different host species (e.g., rabbit polyclonal and mouse monoclonal) and pairing them with spectrally distinct fluorophores, researchers can simultaneously visualize ATL1 alongside other neurological markers. This approach is particularly valuable for examining ATL1's colocalization with endoplasmic reticulum markers or other proteins implicated in hereditary spastic paraplegia.

• Sequential Immunostaining: For complex multi-parameter analyses, sequential rounds of staining, imaging, and antibody stripping can be employed, with ATL1 detection in either the first or subsequent rounds depending on epitope sensitivity to stripping procedures.

• Mass Cytometry (CyTOF): Metal-conjugated ATL1 antibodies can be incorporated into mass cytometry panels for highly multiplexed protein detection in single cells, though this requires specialized conjugation and validation.

• Combined Protein-Nucleic Acid Detection: Protocols integrating ATL1 immunodetection with in situ hybridization enable correlation between ATL1 protein expression and mRNA levels of related genes, providing insights into regulatory mechanisms.

When designing such multi-parameter analyses, researchers should carefully validate that antibody performance is not compromised by multiplexing procedures, particularly regarding potential cross-reactivity between detection systems.

What considerations are important when using ATL1 antibodies in correlation with functional GTPase activity assays?

When correlating ATL1 protein detection with functional GTPase activity, researchers should consider several methodological aspects:

• Epitope Selection: Antibodies targeting regions distant from the GTPase domain may be preferred when studying GTPase activity, as they are less likely to interfere with enzyme function during activity measurements.

• Native Conformation Preservation: For studies correlating protein levels with activity, immunoprecipitation protocols should maintain native protein conformations rather than using denaturing conditions.

• Post-translational Modification Awareness: Since ATL1's GTPase activity can be inhibited by ubiquitination through E3 ligase SYVN1 , researchers should consider using antibodies that can distinguish between modified and unmodified forms or employ parallel detection methods for ubiquitination status.

• Experimental Design for Causality: To establish whether changes in ATL1 levels directly impact GTPase activity, complementary approaches using recombinant proteins or genetic manipulations should supplement antibody-based detection.

• Functional Domain Mutants: When studying how mutations affect function, antibodies recognizing regions outside the mutation site should be selected to ensure detection of both wild-type and mutant proteins with equal efficiency.

This integrated approach combining protein detection and functional assessment provides deeper insights into how alterations in ATL1 contribute to pathologies like hereditary spastic paraplegia.