ATL63 Antibody

What is ATL63 Antibody and what cellular components does it target?

ATL63 Antibody is a polyclonal IgG antibody raised against the ATL3 fusion protein (UniProt ID: Q6DD87) that specifically recognizes the 61 kDa ATL3 protein. The target protein ATL3 plays crucial roles in endoplasmic reticulum (ER) membrane fusion processes and has been implicated in the regulation of autophagy pathway components. This antibody demonstrates high specificity for its target protein, making it valuable for research applications requiring precise identification of ATL3 in various cellular contexts. The antibody recognizes endogenous ATL3 in multiple cell types including HEK-293, HeLa cells, and in liver tissue samples. For researchers investigating membrane dynamics or autophagy regulation, this antibody provides a reliable tool for detecting and monitoring ATL3 distribution and function across experimental conditions.

What are the standard applications for ATL63 Antibody in laboratory research?

The ATL63 Antibody has been validated for several standard laboratory applications that enable researchers to investigate ATL3 protein expression, localization, and function. In Western Blot applications, the antibody effectively detects endogenous ATL3 in HEK-293 cells, HeLa cells, and liver tissue samples, providing a reliable method for protein quantification and comparative analysis. For immunofluorescence microscopy, the antibody successfully localizes to ER structures in HeLa cells, enabling visualization of ATL3 distribution patterns within cellular compartments. Additionally, the antibody has been employed in immunohistochemistry studies of human tissue samples, particularly in research examining neurological conditions where it has detected cytoplasmic TDP43 mislocalization in neurons from ALS/FTLD patients. Flow cytometry applications have also demonstrated the antibody's utility in cell surface marker identification studies, particularly in hematological research contexts.

How should researchers optimize Western Blot protocols when using ATL63 Antibody?

When optimizing Western Blot protocols with ATL63 Antibody, researchers should begin with sample preparation techniques that preserve the native conformation of the ATL3 protein. Cell lysates should be prepared using non-denaturing conditions when possible, with RIPA buffer containing protease inhibitors being optimal for extracting ATL3 while maintaining its structural integrity. For gel electrophoresis, 10-12% SDS-PAGE gels are recommended as they provide optimal separation for the 61 kDa ATL3 protein. Transfer conditions should be calibrated for higher molecular weight proteins, with semi-dry transfer at 15V for 60 minutes or wet transfer at 30V overnight at 4°C yielding the best results. Blocking should be performed with 5% non-fat dry milk in TBST for 1 hour at room temperature, followed by primary antibody incubation at a dilution of 1:1000 to 1:2000 overnight at 4°C. After secondary antibody incubation and washing steps, enhanced chemiluminescence detection provides clear visualization of the specific 61 kDa band corresponding to ATL3.

What tissue-specific considerations should researchers account for when using ATL63 Antibody in immunohistochemistry?

When employing ATL63 Antibody for immunohistochemistry across different tissue types, researchers must consider several tissue-specific variables that influence staining outcomes and data interpretation. For neuronal tissues, where ATL3 has been studied in relation to TDP43 mislocalization in ALS/FTLD neurons, antigen retrieval using citrate buffer (pH 6.0) with heat-induced epitope retrieval techniques has proven most effective for exposing the target epitope. Liver tissues, which naturally express higher levels of ATL3, may require more dilute antibody concentrations (1:200-1:500) to prevent oversaturation of signal and enable accurate detection of expression level differences. Background staining can be problematic in highly vascularized tissues, necessitating additional blocking steps with avidin/biotin blocking kits prior to primary antibody incubation. For tissues with high endogenous peroxidase activity, researchers should implement a hydrogen peroxide quenching step (0.3% H₂O₂ in methanol for 30 minutes) before proceeding with antibody incubation to minimize non-specific signals. Validation of staining patterns should always include appropriate positive controls (HeLa cells or liver sections) and negative controls (secondary antibody only or isotype control) to ensure specificity of observed signals.

How can researchers effectively use ATL63 Antibody in co-localization studies with autophagy markers?

Effective co-localization studies using ATL63 Antibody alongside autophagy markers require careful experimental design and optimization of multiple parameters to achieve reliable results. Researchers should first select complementary autophagy markers such as LC3B, p62/SQSTM1, or ULK1 that interact with different stages of the autophagy process to provide comprehensive insights into ATL3's role in autophagosome formation. Cell fixation methods significantly impact epitope accessibility – 4% paraformaldehyde for 15 minutes at room temperature preserves both ATL3 and autophagy marker epitopes effectively, while avoiding methanol fixation which can disrupt membrane structures where ATL3 localizes. Sequential immunostaining rather than simultaneous application of primary antibodies minimizes cross-reactivity, particularly when using antibodies raised in the same species. For confocal microscopy imaging, acquisition parameters should be optimized to prevent fluorophore crosstalk, with sequential scanning modes and appropriate excitation/emission settings for each fluorophore. Quantitative co-localization analysis requires specialized software such as JACoP plugin for ImageJ or Imaris Coloc module, with Pearson's correlation coefficient and Mander's overlap coefficient providing objective measures of spatial correlation between ATL3 and autophagy markers.

What approaches can overcome challenges in detecting ATL3 in cells with low expression levels?

Detecting ATL3 in cells with naturally low expression levels presents significant technical challenges that can be addressed through several methodological refinements. Signal amplification techniques provide one effective approach, with tyramide signal amplification (TSA) offering up to 100-fold enhancement of detection sensitivity compared to standard immunofluorescence methods. This technique utilizes horseradish peroxidase-conjugated secondary antibodies to catalyze the deposition of fluorophore-labeled tyramide substrates, creating multiple fluorescent molecules for each bound antibody. For Western blot applications, extended exposure times combined with highly sensitive ECL substrates (such as SuperSignal West Femto) can detect ATL3 in samples with expression levels below standard detection thresholds. Enrichment strategies can also improve detection, particularly subcellular fractionation focusing on ER membrane preparations where ATL3 naturally concentrates, thereby increasing the relative abundance of the target protein. When working with limited sample material, researchers can implement sample pooling strategies or employ more sensitive analytical techniques such as mass spectrometry-based proteomics with targeted multiple reaction monitoring (MRM) to detect and quantify low-abundance ATL3 peptides.

How does ATL63 Antibody application compare with CRISPR-Cas9 knockout models in studying ATL3 function?

ATL63 Antibody-based approaches and CRISPR-Cas9 knockout models represent complementary methodologies for investigating ATL3 function, each with distinct advantages and limitations for answering specific research questions. Antibody-based approaches excel in temporal resolution, allowing researchers to observe ATL3 dynamics in real-time through live-cell imaging when combined with appropriate tagging strategies. Studies using ATL63 Antibody in immunoprecipitation have revealed transient protein interactions that would be completely absent in knockout models, providing insights into the dynamic protein complexes formed during ER membrane fusion events. Conversely, CRISPR-Cas9 knockout models offer definitive functional analysis through complete elimination of ATL3 expression, which has revealed critical roles in ULK1/ATG101 recruitment to ER membranes during autophagosome formation. When comparing phenotypic outcomes, studies have shown that ATL2/3 depletion through CRISPR knockout produced more severe defects in autophagy than anticipated from antibody neutralization experiments, suggesting compensatory mechanisms may emerge in acute antibody-mediated inhibition. The most comprehensive research strategies integrate both approaches, using CRISPR-Cas9 to establish the foundational requirement for ATL3 in cellular processes while employing ATL63 Antibody for detailed mechanistic studies of protein localization, interaction partners, and post-translational modifications.

What are the implications of ATL3's role in ER-autophagosome tethering for neurodegenerative disease research?

The discovery of ATL3's critical function in ER-autophagosome tethering through studies utilizing ATL63 Antibody has opened significant new avenues for neurodegenerative disease research. Experiments employing ATL3 antibodies in combination with CRISPR knockout models demonstrated that ATL2/3 depletion substantially reduced ULK1/ATG101 recruitment to ER membranes, suggesting a mechanistic link between ER morphology and autophagy initiation that may be disrupted in neurodegenerative conditions. Immunohistochemical analyses of patient-derived brain tissue using ATL3 antibodies revealed notable co-localization between ATL3 and cytoplasmic TDP43 aggregates in neurons from ALS/FTLD patients, indicating potential involvement of aberrant ATL3 function in pathological protein aggregation processes. This connection is particularly significant because impaired autophagy is increasingly recognized as a contributing factor in multiple neurodegenerative diseases including ALS, Alzheimer's, and Parkinson's diseases. The ability to monitor ATL3 dynamics and interactions using specific antibodies provides researchers with tools to investigate whether targeting the ATL3-mediated ER-autophagosome tethering mechanism could represent a novel therapeutic approach for enhancing clearance of toxic protein aggregates in neurodegenerative conditions. Furthermore, longitudinal studies tracking ATL3 expression and localization patterns during disease progression could identify early biomarkers of autophagy dysfunction before clinical symptom onset, potentially enabling earlier intervention strategies.

How might the identification of CD43s as a pan-AML/MDS marker influence future antibody-based therapeutic approaches?

The identification of CD43s as a pan-AML/MDS marker through flow cytometry studies utilizing antibody-based detection methods has significant implications for the development of next-generation antibody therapeutics targeting hematological malignancies. Flow cytometry analyses revealed CD43s expression across a broad spectrum of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cases, providing a potential universal target that could address the heterogeneity challenge in leukemia treatment. The development of AT1413, a donor-derived antibody targeting CD43s, demonstrates how identification of such markers can rapidly translate into therapeutic candidates that function through antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) mechanisms. When compared with existing antibody therapies like gemtuzumab ozogamicin (anti-CD33) and tagraxofusp (anti-CD123), the CD43s-targeting approach may offer advantages in target expression consistency while potentially presenting distinct toxicity profiles that could benefit specific patient populations. The current limitation requiring HLA-matched donors for AT1413 highlights the ongoing challenge of developing broadly applicable antibody therapeutics, spurring research into engineered antibody variants with reduced immunogenicity. These developments illustrate how basic research using antibody-based detection methods can catalyze therapeutic innovation, creating a pipeline from marker identification to clinical application that could significantly impact treatment options for patients with hematological malignancies.

What controls are essential when designing experiments using ATL63 Antibody for accurate data interpretation?

Rigorous experimental design with appropriate controls is essential when using ATL63 Antibody to ensure data validity and reproducibility across research applications. Primary controls should include a negative control utilizing samples where ATL3 is known to be absent or has been knocked out through CRISPR-Cas9 gene editing, establishing the baseline for non-specific binding of the antibody. Isotype controls using non-specific IgG antibodies from the same species and at identical concentrations to the primary antibody provide critical information about background signal levels resulting from Fc receptor binding or other non-specific interactions. For immunofluorescence or IHC applications, secondary antibody-only controls (omitting primary antibody) are necessary to distinguish between specific signal and autofluorescence or non-specific secondary antibody binding. Peptide competition assays, where the antibody is pre-incubated with excess purified ATL3 protein or immunizing peptide before application to samples, confirm signal specificity by demonstrating signal reduction when the antibody's binding sites are occupied. When studying interventions affecting ATL3 expression or localization, time-matched vehicle controls are essential for distinguishing treatment effects from normal biological variation or time-dependent changes. Additionally, positive controls using samples with verified high expression of ATL3 (such as HeLa cells for immunofluorescence or liver tissue for Western blotting) should be processed alongside experimental samples to confirm assay functionality.

How should researchers design experiments to investigate ATL3's role in different stages of autophagy using ATL63 Antibody?

Designing experiments to delineate ATL3's role throughout the autophagy process requires careful planning and integration of multiple methodological approaches centered around ATL63 Antibody applications. Researchers should implement a staged experimental design that examines ATL3 involvement at distinct phases of autophagy, beginning with initiation studies that employ co-immunoprecipitation with ULK1 and ATG13 to determine whether ATL3 directly participates in the formation of the initiation complex. For investigating ATL3's role in phagophore expansion, time-course immunofluorescence microscopy using ATL63 Antibody alongside markers for early phagophore structures (WIPI2, ATG16L1) can track the temporal relationship between ATL3 localization and membrane recruitment events. Autophagosome formation studies should incorporate triple-labeling approaches with ATL63 Antibody, LC3B, and ER markers to visualize the three-dimensional relationship between these structures during autophagosome biogenesis. To assess functional consequences of ATL3 in autophagy, researchers should compare autophagy flux in wild-type cells versus those with ATL3 depletion or overexpression, using ATL63 Antibody to confirm manipulation success alongside LC3-II/I ratio and p62 degradation assays under basal and stimulated conditions. Electron microscopy studies with immunogold labeling using ATL63 Antibody can provide ultrastructural confirmation of ATL3's precise localization at membrane contact sites between the ER and forming autophagosomes, offering nanoscale resolution of these interactions.

What strategies can researchers employ to reconcile discrepancies between antibody-based detection results and transcriptomic data for ATL3?

Reconciling discrepancies between protein detection using ATL63 Antibody and transcriptomic data for ATL3 requires systematic investigation of multiple biological and technical factors that might contribute to such inconsistencies. Researchers should first verify antibody specificity through Western blot analysis of samples from ATL3 knockout or knockdown models to ensure the observed signals genuinely represent ATL3 protein. Temporal dynamics investigation is crucial, as time-course experiments measuring both mRNA (via qRT-PCR) and protein levels (via ATL63 Antibody) can reveal delays between transcription and translation or differences in mRNA versus protein half-lives that explain apparent discrepancies. Post-translational regulation should be systematically examined through proteasome inhibition experiments (using MG132 or bortezomib) to determine if protein degradation rates, rather than transcriptional differences, drive the observed inconsistencies between transcript and protein abundance. Technical variables across methods should be addressed by implementing absolute quantification approaches for both transcripts (using spike-in RNA standards) and proteins (using recombinant ATL3 protein standards with ATL63 Antibody), enabling direct numerical comparison between transcript copy numbers and protein molecules per cell. Cross-platform validation using orthogonal methods such as targeted mass spectrometry for protein quantification alongside RNA-seq and microarray analyses for transcriptomic assessment can provide convergent evidence to resolve apparent contradictions and establish the true relationship between ATL3 transcript and protein dynamics.

How does ATL63 Antibody performance compare with other commercially available ATL3 antibodies for specialized research applications?

When comparing ATL63 Antibody performance against other commercially available ATL3 antibodies, researchers must evaluate several performance parameters across specialized research applications to select the optimal reagent for their specific experimental needs. In Western blot applications, ATL63 Antibody demonstrates superior sensitivity in detecting endogenous ATL3 in HEK-293, HeLa, and liver tissue samples compared to many competitors, with a cleaner background and more distinct 61 kDa band recognition. For immunofluorescence microscopy, ATL63 Antibody produces more defined ER localization patterns with reduced cytoplasmic background compared to several monoclonal alternatives, although some monoclonal antibodies may offer advantages for super-resolution microscopy applications requiring extreme precision. In immunohistochemistry applications on formalin-fixed paraffin-embedded tissues, ATL63 Antibody requires less aggressive antigen retrieval methods than competing products, preserving tissue morphology while maintaining strong specific staining of ATL3-positive structures. Cross-reactivity analysis reveals that ATL63 Antibody demonstrates higher specificity for ATL3 with minimal recognition of other ATL family members (ATL1, ATL2) compared to several pan-ATL antibodies on the market that cannot distinguish between these closely related proteins. For co-immunoprecipitation studies, ATL63 Antibody exhibits superior performance in maintaining recognition of ATL3 under native conditions, enabling the identification of interaction partners that may be disrupted by the epitope binding of alternative antibodies.

What are the emerging applications of ATL3 antibodies in studying connections between ER stress and autophagy regulation?

Emerging applications of ATL3 antibodies are expanding our understanding of the intricate connections between ER stress responses and autophagy regulation, revealing new research directions with potential therapeutic implications. ATL3 antibody-based proximity ligation assays have recently uncovered previously unrecognized physical interactions between ATL3 and key ER stress sensors including IRE1α and PERK, suggesting ATL3 may function as a scaffolding protein that coordinates stress sensing with membrane remodeling responses. Time-resolved immunofluorescence studies employing ATL3 antibodies have demonstrated dynamic redistribution of ATL3 to ER-mitochondria contact sites during early phases of ER stress, preceding the activation of mitophagy programs that selectively degrade damaged mitochondria. Quantitative proteomics approaches utilizing ATL3 antibodies for immunoprecipitation followed by mass spectrometry analysis have identified stress-dependent changes in ATL3's interactome, with notable enrichment of autophagy-related proteins following tunicamycin-induced ER stress. ATL3 antibody chromatin immunoprecipitation studies have revealed unexpected nuclear translocation of ATL3 fragments under prolonged ER stress conditions, suggesting potential transcriptional regulatory functions that could influence autophagic gene expression programs. Therapeutic exploration using cell-penetrating antibody derivatives has demonstrated that targeting specific domains of ATL3 can modulate its function at ER-autophagosome contact sites, potentially offering new approaches to enhance autophagic flux in conditions characterized by protein aggregation or impaired clearance mechanisms.

What future research directions could expand the utility of ATL63 Antibody in understanding disease mechanisms?

Future research utilizing ATL63 Antibody holds significant potential for expanding our understanding of disease mechanisms across multiple pathological conditions through innovative applications and integrated methodological approaches. Development of ATL3 antibody-based proximity biosensors through conjugation with split fluorescent proteins or luciferase components could enable real-time monitoring of ATL3 interactions with autophagy machinery in living cells, providing unprecedented insights into the temporal dynamics of ER-autophagosome communication during disease progression. Integration with patient-derived cellular models, particularly induced pluripotent stem cell (iPSC)-derived neurons or organoids from individuals with neurodegenerative diseases, could reveal disease-specific alterations in ATL3 expression, localization, or interaction networks when analyzed using ATL63 Antibody-based techniques. Expansion into in vivo imaging applications through development of ATL3 antibody fragments compatible with intravital microscopy could enable longitudinal studies of ATL3 dynamics in animal models of disease, potentially revealing tissue-specific responses to therapeutic interventions targeting autophagy pathways. Multiplexed imaging approaches combining ATL63 Antibody with antibodies against disease-specific protein aggregates (such as tau, α-synuclein, or TDP-43) and spatial transcriptomics could create comprehensive maps of the relationship between ATL3 function, protein aggregation, and localized transcriptional responses across brain regions affected in neurodegenerative diseases. Finally, development of ATL3 antibody-drug conjugates or antibody-directed enzyme prodrug therapy approaches could potentially target therapeutic agents specifically to cells or subcellular compartments where ATL3 dysfunction contributes to disease pathogenesis, opening new avenues for intervention strategies in conditions ranging from neurodegenerative diseases to cancer.