ATG101 Antibody, FITC conjugated

What is ATG101 and why is it important in autophagy research?

ATG101 is an essential autophagy-related protein that forms part of the ULK1 complex, a critical initiator of the autophagy process. It functions by stabilizing ATG13 and facilitating ULK1 activity, which is necessary for autophagosome formation. In research, ATG101 serves as an important marker for monitoring autophagy initiation and progression . Studies in model organisms like Drosophila have shown that ATG101 mutants accumulate ubiquitin and Ref(2)p (p62 homolog) punctate structures in the brain, suggesting the formation of protein aggregates in the central nervous system when autophagy is impaired . The protein has a molecular weight of approximately 25 kDa and is known to undergo phosphorylation at Ser11 by ULK1, which regulates its function . ATG101 antibodies are therefore valuable tools for investigating fundamental autophagy mechanisms in both normal physiology and disease states.

How does FITC conjugation enhance the utility of ATG101 antibodies in research applications?

FITC (Fluorescein isothiocyanate) conjugation to ATG101 antibodies provides direct visualization capabilities without requiring secondary antibody detection steps. This modification offers several methodological advantages in research:

• Signal amplification efficiency: Direct conjugation eliminates signal loss that can occur with multiple incubation and washing steps in indirect detection methods.

• Multiparameter analysis compatibility: FITC emits in the green spectrum (peak ~525 nm), allowing combination with other fluorophores for co-localization studies.

• Time efficiency: Experiments can be completed more rapidly due to fewer incubation steps.

• Reduced background: Elimination of secondary antibodies reduces potential cross-reactivity issues.

For ATG101 research specifically, FITC-conjugated antibodies allow direct visualization of ATG101 distribution in cellular compartments and its co-localization with other autophagy proteins. When studying phosphorylated ATG101 (Ser11), fluorescence intensity can serve as a quantitative measure of ULK1 kinase activity in response to autophagy induction stimuli .

What are the optimal fixation and permeabilization methods for ATG101 immunofluorescence staining?

The optimization of fixation and permeabilization protocols is critical for preserving ATG101 epitopes while allowing antibody accessibility. Based on experimental evidence:

For phosphorylated ATG101 detection, methanol fixation is generally preferred as it better preserves phosphoepitopes. When conducting co-localization studies with other autophagy markers, it's essential to verify that the chosen fixation method works well for all target proteins. For instance, when studying ATG101 localization relative to LC3-positive autophagosomes, a brief fixation with 4% PFA followed by methanol post-fixation often yields optimal results for both markers.

For tissues samples, additional optimization may be necessary, as demonstrated in Drosophila brain studies where protein aggregate accumulation was successfully visualized in ATG101 mutants .

What are the critical controls needed when using ATG101 antibody for autophagy studies?

Rigorous experimental controls are essential for valid interpretation of ATG101 antibody staining results:

• Specificity controls:

  • Transfected-only validation (as indicated for phospho-ATG101 antibodies)

  • ATG101 knockdown/knockout cells as negative controls

  • Antibody pre-absorption with immunizing peptide

• Biological controls:

  • Comparison between basal and autophagy-induced conditions (e.g., starvation, rapamycin)

  • ULK1 inhibitor treatment to prevent Ser11 phosphorylation

  • Comparison with other autophagy markers (LC3, WIPI2)

• Technical controls:

  • Secondary antibody-only controls for indirect detection

  • Isotype controls matched to the ATG101 antibody

  • Fluorescence minus one (FMO) controls for multicolor flow cytometry

When investigating ATG101 in disease models, additional controls should include comparison with appropriate wild-type samples and verification of autophagy status using independent methods such as transmission electron microscopy or LC3-II/I ratio analysis by western blot.

How can ATG101 antibodies be used to investigate the crosstalk between autophagy and immune signaling pathways?

ATG101 antibodies provide valuable tools for investigating the increasingly recognized connections between autophagy and immune regulation:

• Dual immunofluorescence approaches: FITC-conjugated ATG101 antibodies can be combined with markers of immune signaling pathways (e.g., NF-κB, STAT proteins) to visualize potential co-regulation. This approach revealed that in certain contexts, ATG101-containing complexes relocalize to immune synapses during T cell activation.

• Proximity ligation assays: Using ATG101 antibodies in PLA experiments can detect direct protein-protein interactions between ATG101 and immune regulatory proteins, providing spatial resolution below 40nm.

• ChIP-seq applications: Some autophagy proteins, including components of the ULK1 complex, have been implicated in transcriptional regulation. ATG101 antibodies can be utilized in ChIP-seq experiments to investigate potential nuclear roles.

Recent research has particularly highlighted connections between autophagy and immune checkpoint regulation. While distinct from the autophagy protein ATG101, the bispecific antibody ATG-101 provides interesting insights into autophagy-immune connections. ATG-101 targets both PD-L1 and 4-1BB, demonstrating how modulation of immune checkpoints can overcome resistance to immune checkpoint inhibitors . This research direction suggests that investigating potential crosstalk between ATG101-mediated autophagy and immune checkpoint molecules could be a fruitful area for future research.

What methodological approaches allow for the quantitative assessment of ATG101 dynamics during different phases of autophagy?

Quantitative assessment of ATG101 dynamics requires sophisticated methodological approaches:

For the most comprehensive analysis, researchers should combine multiple techniques. For example, flow cytometry with phospho-ATG101 (Ser11) antibodies can quantify ULK1 activation across cell populations, while live-cell imaging provides insights into the spatiotemporal dynamics of ATG101 recruitment to pre-autophagosomal structures.

The phosphorylation state of ATG101 at Ser11 serves as a particularly useful quantitative marker, as demonstrated by studies using phospho-specific antibodies . This phosphorylation event, catalyzed by ULK1, represents an early step in autophagy initiation and can be monitored over time to track autophagy progression.

How can researchers address non-specific binding issues with ATG101 antibodies in immunofluorescence applications?

Non-specific binding is a common challenge when working with antibodies, including those targeting ATG101:

• Optimization strategies for reducing background:

  • Increase blocking stringency (5-10% normal serum from species unrelated to primary and secondary antibodies)

  • Include 0.1-0.3% Triton X-100 in antibody diluent to reduce hydrophobic interactions

  • Add 0.1-0.5M NaCl to antibody diluent to disrupt low-affinity electrostatic interactions

  • For FITC-conjugated antibodies specifically, addition of 0.01-0.1% sodium azide helps prevent photobleaching

• Protocol modifications for problematic samples:

  • For tissues with high autofluorescence, consider Sudan Black B treatment (0.1-0.3% in 70% ethanol)

  • Pre-absorption of antibody with tissue lysate from ATG101-knockout samples

  • For neuronal tissues showing high background (as observed in Drosophila studies ), extend blocking time and use detergent-containing wash buffers

• Signal validation approaches:

  • Compare staining patterns between multiple antibodies targeting different epitopes of ATG101

  • Verify subcellular localization patterns against published data

  • Confirm specificity using genetic knockdown/knockout models

When possible, researchers should validate that their antibody recognizes only the intended target in their experimental system, as antibody reactivity may vary between applications (Western blot vs. immunofluorescence) and between species, as noted in product information for commercial ATG101 antibodies .

How should researchers interpret changes in ATG101 expression or localization in the context of disease models?

Interpreting ATG101 data in disease contexts requires careful consideration of multiple factors:

• Expression level interpretation:

  • Increased ATG101 expression often indicates compensatory upregulation of autophagy in response to stress

  • Decreased expression may suggest autophagy suppression or pathological disruption

  • Changes should be correlated with other autophagy markers (LC3-II, p62) to distinguish between induction and blockade

• Localization pattern analysis:

  • Punctate redistribution typically indicates activation and recruitment to pre-autophagosomal structures

  • Abnormal aggregation may indicate protein misfolding or autophagy dysfunction

  • Nuclear localization has been reported in some contexts and may indicate non-canonical functions

• Phosphorylation state assessment:

  • Increased phospho-ATG101 (Ser11) without corresponding autophagy activation may indicate pathway dysregulation

  • Phosphorylation patterns should be evaluated in relation to disease progression

The interpretation becomes particularly complex in neurodegenerative diseases where protein aggregation is prominent. Studies in Drosophila have shown that ATG101 mutations lead to accumulation of ubiquitinated proteins and Ref(2)p (p62 homolog) in the brain , suggesting that ATG101 dysfunction contributes to the protein aggregation phenotype characteristic of many neurodegenerative conditions. When evaluating potential disease biomarkers, changes in ATG101 should be considered within the broader context of autophagy dysfunction and not as isolated phenomena.

How are ATG101 antibodies being utilized to investigate the role of selective autophagy in cancer and neurodegenerative diseases?

ATG101 antibodies are increasingly employed to investigate selective autophagy in various disease contexts:

• Cancer research applications:

  • Evaluating ATG101 expression correlations with treatment resistance

  • Investigating changes in mitophagy (selective autophagy of mitochondria) via co-localization studies

  • Assessing ATG101 interactions with tumor suppressor pathways

• Neurodegenerative disease investigations:

  • Visualization of ATG101 recruitment to protein aggregates in models of Alzheimer's, Parkinson's, and Huntington's diseases

  • Analysis of ATG101 phosphorylation status as a marker of ULK1 activity in brain tissues

  • Monitoring ATG101-dependent selective autophagy of disease-relevant substrates

Studies in Drosophila have demonstrated that ATG101 mutations result in the accumulation of ubiquitinated proteins in the central nervous system , suggesting important roles in neuronal proteostasis. This finding highlights the potential of ATG101 as a target for therapeutic interventions in neurodegenerative diseases characterized by protein aggregation.

In cancer research, interesting parallels can be drawn from studies on the bispecific antibody ATG-101 (distinct from the autophagy protein). ATG-101 activates anti-tumor immunity by targeting PD-L1 and the costimulatory receptor 4-1BB, demonstrating effectiveness against tumors resistant to immune checkpoint inhibitors . This raises intriguing questions about potential connections between autophagy regulation and immune surveillance mechanisms in cancer.

What novel methodological approaches are being developed to study ATG101 interactions with the broader autophagy machinery?

Cutting-edge methodological approaches are expanding our understanding of ATG101's role in autophagy:

• Advanced imaging technologies:

  • Super-resolution microscopy (PALM/STORM) to visualize ATG101 within autophagy initiation complexes at nanoscale resolution

  • Lattice light-sheet microscopy for long-term 3D imaging of ATG101 dynamics with reduced phototoxicity

  • Correlative light and electron microscopy (CLEM) to position ATG101 within the ultrastructural context of forming autophagosomes

• Proteomics and interactomics:

  • Cross-linking mass spectrometry (XL-MS) to capture transient ATG101 protein interactions

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map conformational changes upon complex formation

  • Thermal shift assays to identify compounds that stabilize or destabilize ATG101 interactions

• Functional genomics approaches:

  • CRISPR-Cas9 base editing to introduce specific point mutations in ATG101 (including the Ser11 phosphorylation site)

  • Domain-focused CRISPR screening to identify functional regions of ATG101

  • Conditional degradation systems for temporal control of ATG101 depletion

A particularly promising direction involves the development of biosensors based on ATG101 and its interactions. These include FRET-based reporters that can monitor ATG101-ATG13 interactions in real-time and split-luciferase complementation systems that report on ULK1 complex assembly. Such tools enable dynamic measurement of autophagy initiation in living cells and tissues, providing unprecedented temporal resolution of autophagy regulation.

The development of phospho-specific antibodies against ATG101 modifications, such as the Ser11 phosphorylation by ULK1 , represents another important methodological advance that allows researchers to monitor specific regulatory events in the autophagy initiation cascade.