ATL5 Antibody

What is ATL5 and what are the different contexts in which it appears in research?

ATL5 appears in multiple research contexts that must be clearly distinguished:

In plants, ATL5 (ARABIDOPSIS TÓXICOS EN LEVADURA 5) functions as an E3 ligase that positively regulates seed longevity through mediating protein degradation. It specifically promotes the polyubiquitination and degradation of the ABT1 (ACTIVATOR OF BASAL TRANSCRIPTION 1) protein posttranslationally in Arabidopsis . This plant-specific ATL5 is highly expressed in seed embryos and its expression can be induced during accelerated aging processes.

In human research, ATL5 often refers to the protein product of the ADAMTSL5 gene (ADAMTS Like 5), which is a protein-coding gene. The ADAMTSL5 gene has been associated with several medical conditions including Syndromic X-Linked Intellectual Disability 94 . When discussing human ATL5 antibodies, researchers are typically referring to antibodies that target this protein.

It's crucial for researchers to specify which ATL5 they're investigating, as methodologies and applications differ significantly between plant and human contexts.

What are the primary applications of ATL5 antibodies in research settings?

ATL5 antibodies serve multiple research purposes depending on the specific ATL5 variant being studied:

For plant ATL5 research, antibodies are essential for tracking E3 ligase activity, detecting protein-protein interactions (particularly with substrates like ABT1), and monitoring ubiquitination processes. These antibodies enable researchers to investigate the regulatory mechanisms of seed longevity and protein degradation pathways .

For human ADAMTSL5 research, antibodies are applied in immunocytochemistry-immunofluorescence (ICC-IF) techniques as validated by enhanced validation protocols . These antibodies help detect and localize the protein in various cellular contexts, supporting research on associated medical conditions.

In both contexts, ATL5 antibodies facilitate protein detection in techniques such as Western blotting, immunoprecipitation, immunohistochemistry, and fluorescence microscopy. The specific application determines the optimal antibody format and validation requirements.

How should researchers validate the specificity of ATL5 antibodies?

Validating ATL5 antibody specificity requires a multi-faceted approach:

First, researchers should perform Western blot analysis using positive and negative control samples. For human ADAMTSL5 antibodies, comparing tissues with known expression levels of the target protein against knockout or knockdown samples provides crucial specificity data . The antibody should recognize a band of the expected molecular weight with minimal non-specific binding.

Second, implement parallel validation using orthogonal techniques. For instance, if studying human ATL5, perform both tissue-based assays (TBA) and cell-based assays (CBA) for confirmatory purposes, similar to the approach used in validating other antibodies targeting intracellular proteins .

Third, include genetic controls where feasible. For plant ATL5 studies, comparing wild-type Arabidopsis samples with atl5-2 mutants allows verification of antibody specificity . Similarly, CRISPR/Cas9-mediated knockout or siRNA knockdown models provide valuable controls for human ADAMTSL5 studies.

Finally, cross-reactivity testing against related proteins (particularly other ADAMTS family members for human studies or related E3 ligases for plant studies) ensures the antibody exclusively recognizes the intended target.

What are the optimal experimental conditions for using ATL5 antibodies in co-immunoprecipitation studies?

When using ATL5 antibodies for co-immunoprecipitation (co-IP) to study protein interactions:

For plant ATL5 studies investigating E3 ligase-substrate interactions (such as ATL5-ABT1), researchers should:

• Extract proteins under non-denaturing conditions using buffers containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% NP-40, and protease inhibitor cocktail

• Include 10-20 mM N-ethylmaleimide to preserve ubiquitination states

• Pre-clear lysates using protein A/G beads before immunoprecipitation to reduce non-specific binding

• Confirm interactions through both forward and reverse co-IP approaches (precipitating with anti-ATL5 and anti-substrate antibodies respectively)

For human ADAMTSL5 studies:

• Use RIPA buffer with lower detergent concentrations (0.1% SDS, 0.5% sodium deoxycholate) for membrane-associated protein preservation

• Consider crosslinking approaches for transient interactions

• Validate results with recombinant expression systems for confirmation

Both contexts benefit from:

• Titrating antibody concentrations (typically 2-5 μg per mg of protein lysate)

• Including appropriate negative controls (non-specific IgG of the same species)

• Confirming results using reciprocal co-IP where possible

How can ATL5 antibodies be integrated with proteomic approaches to identify novel interaction partners?

Integrating ATL5 antibodies with proteomics offers powerful approaches for discovering novel interaction networks:

For plant ATL5 E3 ligase research, implement immunoprecipitation coupled with mass spectrometry (IP-MS) to identify potential substrates beyond ABT1. This approach has been successfully used to characterize E3 ligase networks in Arabidopsis . Extract proteins from seeds under various aging conditions, perform IP with ATL5 antibodies, then analyze precipitated proteins using LC-MS/MS. Compare results with control precipitations to filter out non-specific interactions.

For human ADAMTSL5 studies, proximity-dependent biotin identification (BioID) combined with ATL5 antibody validation offers a complementary approach. Express ATL5-BioID fusion proteins, identify biotinylated proximal proteins via MS, then validate key interactions using conventional co-IP with anti-ATL5 antibodies.

Both research areas benefit from incorporating stable isotope labeling (SILAC or TMT) to quantitatively compare interaction profiles under different conditions. For example, comparing interaction partners in wild-type versus mutant backgrounds (such as atl5-2 plant mutants) can reveal condition-specific interactions .

Data analysis should incorporate bioinformatic filtering using databases of known contaminants and statistical thresholds (typically p < 0.05 and fold-change > 2) to identify high-confidence interactors.

What techniques can distinguish between canonical and non-canonical roles of ATL5 in disease models?

Distinguishing canonical from non-canonical ATL5 functions requires sophisticated approaches similar to those used in other antibody research:

For human ADAMTSL5 studies, researchers can adapt the multi-feature antibody profiling approach used in COVID-19 research, where antibodies against canonical (e.g., spike protein) and non-canonical antigens were analyzed . This approach involves:

In practice, this could involve:

• Developing a panel of antibodies targeting different ATL5 domains

• Examining cellular responses to these antibodies in various disease contexts

• Using machine learning approaches to identify patterns associated with different outcomes

For plant ATL5 research, distinguishing canonical (ubiquitination of ABT1) versus non-canonical functions can be approached by:

• Using domain-specific ATL5 antibodies to track different protein complexes

• Combining with mutational studies targeting specific functional domains

• Employing proximity labeling techniques to identify context-specific interaction partners

How can researchers address inconsistent results when using ATL5 antibodies across different experimental systems?

When facing inconsistent results with ATL5 antibodies across experimental systems:

First, evaluate antibody performance across platforms. Human ADAMTSL5 antibodies validated for ICC-IF may not perform equally in Western blotting or IHC . Validate each application independently and establish optimal conditions for each technique.

Second, consider epitope accessibility issues. For intracellular antigens like ATL5, epitope masking due to protein-protein interactions or post-translational modifications can affect antibody binding. Test multiple antibody clones targeting different epitopes, similar to approaches used for other intracellular antigens like AK5 .

Third, optimize sample preparation for the specific ATL5 variant. Plant ATL5 studies may require plant-specific extraction buffers with appropriate detergents and protease inhibitors tailored to plant tissues .

Fourth, implement biological validation using genetic approaches. For plant studies, complement antibody experiments with atl5-2 mutant comparisons . For human studies, CRISPR knockouts or siRNA approaches provide essential controls.

What are the critical considerations when using ATL5 antibodies in tissue-specific studies?

For tissue-specific studies using ATL5 antibodies:

First, assess baseline expression levels. In plant research, ATL5 is highly expressed in seed embryos but may have tissue-specific expression patterns elsewhere . For human ADAMTSL5, examine reference databases for expression patterns across tissues before designing experiments.

Second, optimize fixation and permeabilization protocols for the specific tissue. For plant embryonic tissues, traditional formaldehyde fixation may be insufficient; consider using different crosslinkers or combining fixatives. For human tissues, balanced fixation that preserves epitope accessibility while maintaining tissue architecture is essential.

Third, implement rigorous controls specific to each tissue type:

• Include tissue from knockout/knockdown models when available

• Use competing peptides to confirm specificity in the tissue context

• Perform parallel detection with orthogonal methods (like in situ hybridization)

Fourth, consider the impact of tissue-specific post-translational modifications. The phosphorylation or ubiquitination status of ATL5 may vary by tissue, affecting antibody recognition. When possible, use phospho-specific or modification-specific antibodies to distinguish modified forms.

Fifth, when studying intracellular proteins like ATL5, consider the approaches used for other intracellular antigens. For instance, in anti-AK5 encephalitis research, a combination of cell-based and tissue-based assays provides superior sensitivity and specificity compared to either method alone .

How might ATL5 antibodies contribute to understanding conserved mechanisms across species?

ATL5 antibodies can illuminate evolutionary conserved mechanisms through comparative studies:

The E3 ligase function of plant ATL5 in regulating protein degradation represents a fundamental cellular mechanism . Applying similar antibody-based techniques to study human ADAMTSL5 could reveal whether functional parallels exist despite sequence divergence.

Research approaches could include:

• Developing antibodies recognizing structurally conserved domains across species

• Comparing interaction networks identified through immunoprecipitation-mass spectrometry in different model organisms

• Investigating whether the regulatory mechanisms identified in plant ATL5 (ubiquitination-mediated protein degradation) have parallels in human ADAMTSL5 function

This cross-species approach resembles strategies used in coronavirus antibody research, where antibodies against conserved viral proteins provided insights into potential pan-coronavirus immunity . Similarly, ATL5 antibodies targeting conserved epitopes could reveal fundamental biological processes maintained across evolutionary distance.

What methodological innovations could enhance the specificity and utility of ATL5 antibodies in complex research applications?

Emerging technologies promise to enhance ATL5 antibody applications:

Single-cell antibody-based proteomics could reveal cell-type-specific ATL5 functions by combining antibody detection with single-cell sequencing. This would be particularly valuable for understanding human ADAMTSL5 in heterogeneous tissues associated with intellectual disability .

Engineered antibody fragments (Fabs, scFvs, nanobodies) against ATL5 could improve penetration in complex tissues and reduce background signal. These smaller binding molecules might access epitopes unavailable to conventional antibodies.

CRISPR-based tagging of endogenous ATL5 combined with anti-tag antibodies could circumvent specificity issues. By introducing small epitope tags into endogenous ATL5 using CRISPR/Cas9, researchers could leverage highly validated tag-specific antibodies while preserving native protein function and expression.

Multiplexed imaging techniques like Imaging Mass Cytometry or CODEX, when adapted for ATL5 detection, would allow simultaneous visualization of ATL5 with dozens of other proteins, revealing complex interaction networks in their native context.