ATG11 Antibody

What is the function of ATG11 protein and why is it important to study with antibodies?

ATG11 serves as a receptor protein for cargo recognition in selective autophagy and is essential for initiating glucose starvation-induced autophagy . Recent research has revealed ATG11 plays additional roles in chromosome transmission and spindle positioning that are independent of its autophagy function . Antibodies against ATG11 are crucial tools for detecting these diverse functions, as they allow researchers to visualize protein localization, quantify expression levels, and identify protein-protein interactions. The importance of ATG11 antibodies is magnified by the discovery that ATG11 undergoes post-translational modifications, particularly phosphorylation by ATG1 kinase, which regulates its function in selective autophagy .

What types of ATG11 antibodies are available for research purposes?

ATG11 antibodies typically fall into several categories:

When selecting an ATG11 antibody, researchers should consider which domain or modification state they are investigating based on research objectives.

How can I validate the specificity of an ATG11 antibody?

Validating ATG11 antibody specificity is critical for generating reliable research results. A comprehensive validation approach should include:

• Genetic control validation: Test the antibody in wild-type versus atg11Δ mutant cells. A specific antibody will show no signal in the knockout samples .

• Recombinant protein control: Use purified recombinant ATG11 as a positive control to verify antibody recognition.

• Immunoprecipitation followed by mass spectrometry: This confirms that the antibody is pulling down ATG11 rather than cross-reactive proteins.

• Tagged protein comparison: Compare detection of endogenous ATG11 with epitope-tagged versions (GFP-ATG11, ATG11-3×HA) using both the ATG11 antibody and tag-specific antibodies .

• Phosphatase treatment: For phospho-specific antibodies, treating samples with phosphatase should eliminate signal if the antibody is truly phospho-specific .

In published studies, researchers have validated ATG11 antibodies using ATG11-3×HA genomic tagging strategies and comparing antibody reactivity under various conditions to confirm specificity .

How can ATG11 antibodies be used to study the protein's phosphorylation state during autophagy?

ATG11 undergoes phosphorylation under various autophagy-inducing conditions including rapamycin treatment, nitrogen starvation, and glucose starvation . To study these modifications:

• Phosphorylation detection: Use standard Western blotting with ATG11 antibodies to observe mobility shifts, which appear as upward band shifts on SDS-PAGE gels .

• Phospho-specific antibodies: For detecting specific phosphorylation sites, phospho-specific antibodies can be developed against known ATG1-mediated phosphorylation sites.

• Phosphatase assays: Treat immunoprecipitated ATG11 with phosphatases and compare to untreated samples to confirm band shifts are due to phosphorylation .

• Kinase dependency analysis: Compare ATG11 phosphorylation in wild-type versus atg1Δ cells or cells expressing kinase-dead (KD) ATG1 mutants. Research has demonstrated that ATG11 phosphorylation is dependent on ATG1 kinase activity and requires ATG13, which is necessary for ATG1 activation .

• Comparative analysis during different stresses: Monitor ATG11 phosphorylation under different autophagy-inducing conditions to identify condition-specific phosphorylation patterns .

This methodology has revealed that ATG1-mediated phosphorylation of ATG11 is a key regulatory mechanism for selective autophagy activation.

What is the optimal protocol for immunoprecipitating ATG11 and its interacting partners?

For successful immunoprecipitation of ATG11 and its binding partners:

• Lysis buffer optimization: Use a buffer containing 50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and protease/phosphatase inhibitor cocktails to preserve protein interactions .

• Cross-linking consideration: For transient interactions, consider using a mild cross-linking agent like DSP (dithiobis(succinimidyl propionate)) before cell lysis.

• Antibody selection: Use antibodies targeting the domain not involved in the protein interaction of interest. For instance, avoid C-terminal antibodies when studying interactions with cargo receptors ATG19 and ATG32, which bind the C-terminal region .

• Co-immunoprecipitation controls: Always include non-specific IgG controls and lysates from atg11Δ strains .

• Detection strategy: For detecting ATG11 interactions with proteins like ATG1, ATG9, ATG29, and ATG31, either use antibodies specific to each protein or use tagged versions of these proteins .

Research has successfully used this approach to identify critical interaction domains, such as the requirement of CC1, CC2, and CC3 domains of ATG11 for association with ATG1, ATG29, and ATG31 during glucose starvation conditions .

How can ATG11 antibodies be used to investigate its non-canonical roles in chromosome transmission?

Recent research has uncovered ATG11's unexpected role in chromosome transmission independent of its autophagy function . To investigate this:

• Subcellular fractionation: Use ATG11 antibodies to detect the protein in nuclear fractions versus cytoplasmic compartments.

• Chromatin immunoprecipitation (ChIP): Apply ATG11 antibodies for ChIP assays to identify potential associations with chromosomal regions.

• Co-localization studies: Combine ATG11 antibodies with markers for spindle pole bodies (SPBs) and microtubules in immunofluorescence studies to visualize ATG11's association with the mitotic machinery. Research has shown ATG11 localizes proximal to the old SPB .

• Comparative analysis in different mutants: Use ATG11 antibodies to compare protein levels and localization patterns in various mitotic checkpoint mutants (mad2Δ, bub2Δ) to elucidate pathway connections .

• Cell cycle synchronization studies: Apply ATG11 antibodies to detect expression and modification changes throughout the cell cycle, particularly during G2/M and anaphase transitions where ATG11 plays critical roles .

This methodology has revealed that ATG11 contributes to spindle positioning dependent on the Kar9 pathway and preserves asymmetric inheritance, providing insights into its non-autophagy functions .

What are the key optimization steps for ATG11 detection by Western blotting?

Optimizing Western blotting for ATG11 detection requires attention to several parameters:

• Sample preparation: When extracting proteins from yeast cells, use glass bead lysis in the presence of protease inhibitors to prevent degradation. For phosphorylated ATG11 detection, include phosphatase inhibitors .

• Gel percentage selection: Use 6-8% SDS-PAGE gels to achieve optimal separation of ATG11 (approximately 135 kDa) and its phosphorylated forms, which exhibit mobility shifts .

• Transfer conditions: For large proteins like ATG11, employ longer transfer times or semi-dry transfer systems with methanol-free buffers to enhance transfer efficiency.

• Blocking optimization: Use 5% non-fat dry milk in TBST for general ATG11 antibodies; for phospho-specific antibodies, use 5% BSA instead as milk contains phosphatases.

• Antibody dilutions and incubation:

  • Primary antibody: 1:1000-1:5000 dilution (optimized for each antibody)

  • Secondary antibody: 1:5000-1:10000 dilution

  • Incubation times: Overnight at 4°C for primary; 1 hour at room temperature for secondary

• Signal detection: For phosphorylated ATG11, which may be less abundant, use high-sensitivity chemiluminescent substrates or fluorescence-based detection systems.

This approach has successfully been used to detect ATG11 modifications under various autophagy-inducing conditions .

What controls should be included when using ATG11 antibodies in experimental designs?

A robust experimental design with ATG11 antibodies should include these controls:

• Genetic controls:

  • atg11Δ strain (negative control)

  • Complemented atg11Δ with wild-type ATG11 (rescue control)

  • Various ATG11 domain mutants for domain-specific functions

• Treatment controls:

  • Untreated versus autophagy induction conditions (rapamycin, nitrogen starvation, glucose starvation)

  • Phosphatase treatment for phosphorylation studies

  • Time-course sampling to capture dynamic changes

• Pathway component controls:

  • atg1Δ and atg13Δ strains to confirm kinase-dependent effects

  • atg5Δ, atg9Δ, and atg14Δ strains as controls for autophagy-dependent but ATG1-independent effects

  • mad2Δ and bub2Δ for spindle checkpoint studies

• Loading and technical controls:

  • Housekeeping proteins (e.g., Pgk1 used at 1:10,000 dilution)

  • Pre-immune serum for polyclonal antibodies

  • Isotype-matched irrelevant antibodies for monoclonals

Including these controls allows for reliable interpretation of ATG11 antibody results across diverse experimental conditions and genetic backgrounds.

How should researchers interpret ATG11 antibody results when studying its dual roles in autophagy and chromosome transmission?

Interpreting ATG11 antibody data requires careful consideration of context:

• Distinguish between roles based on experimental conditions:

  • Autophagy role: Predominant under starvation conditions or rapamycin treatment

  • Chromosome transmission role: More evident during normal growth or at elevated temperatures (37°C)

• Consider protein modifications:

  • Mobility shifts indicate phosphorylation during autophagy induction

  • Different phosphorylation patterns may correlate with distinct functions

• Examine localization patterns:

  • Cytoplasmic puncta formation typically indicates autophagy function

  • Association with spindle pole bodies suggests chromosome transmission role

• Cross-reference with functional assays:

  • Autophagy role: Monitor selective autophagy cargo (e.g., Ape1 processing)

  • Chromosome transmission role: Measure chromosome loss rates, microtubule dynamics, or cell cycle progression

• Genetic interaction interpretation:

  • Synthetic lethality with spindle assembly checkpoint mutants (mad2Δ, bub2Δ) in the presence of microtubule poisons indicates chromosome segregation functions

  • Interaction with autophagy machinery indicates autophagic roles

Understanding that these functions are separable is critical—research has demonstrated that autophagy-deficient cells do not show increased chromosome loss rates, confirming ATG11's non-canonical role is distinct from its autophagy function .

How can researchers address common issues with ATG11 detection by Western blotting?

When troubleshooting ATG11 Western blot problems, consider these solutions:

When working with phosphorylated ATG11, it's particularly important to verify that mobility shifts are indeed due to phosphorylation by treating samples with phosphatases and by comparing wild-type cells with those lacking the relevant kinase (ATG1) or its activator (ATG13) .

What techniques can improve detection of ATG11 protein-protein interactions?

To enhance detection of ATG11 interactions:

• Optimize immunoprecipitation conditions:

  • Buffer composition: Test different detergent types and concentrations (0.1-1% Triton X-100, NP-40, or CHAPS)

  • Salt concentration: Adjust NaCl concentration (100-300 mM) to balance specificity and preservation of interactions

  • Incubation time: Shorter times may preserve transient interactions

• Alternative tagging strategies:

  • Protein A tagging for direct pulldown without antibodies

  • Split-tag approaches for detecting specific interaction pairs

  • Proximity-dependent labeling (BioID, APEX) to capture transient interactions

• Domain-specific approaches:

  • Target specific domains known to mediate interactions (e.g., CC1, CC2, CC3 domains for ATG1, ATG29, ATG31 interactions)

  • Use truncated constructs to map precise interaction regions

• Crosslinking strategies:

  • Reversible crosslinkers (DSP) for preserving interactions through purification

  • Photo-activatable crosslinkers for capturing interactions in live cells

• Complementary techniques:

  • Yeast two-hybrid assays, which have successfully identified ATG11 interaction with ATG9 through the I569 residue in the CC2 domain

  • Fluorescence resonance energy transfer (FRET) for in vivo interaction confirmation

These approaches have enabled researchers to discover critical interactions, such as ATG11's binding to ATG1, ATG29, and ATG31, but not ATG13 and ATG17, under glucose starvation conditions .

How can researchers address contradictory results when comparing ATG11 antibody data across different experimental systems?

When facing contradictory ATG11 antibody results:

• Evaluate antibody specificity differences:

  • Epitope location: Different antibodies may recognize distinct epitopes affected differently by protein conformation or interactions

  • Validation status: Confirm each antibody has been properly validated in the specific experimental system

• Consider post-translational modification status:

  • Phosphorylation state: ATG11 shows different mobility patterns depending on autophagy induction status

  • Other modifications: Ubiquitination or other modifications may affect antibody recognition

• Examine experimental condition variations:

  • Autophagy induction methods: Different starvation protocols or rapamycin concentrations may yield varying results

  • Growth conditions: Temperature affects ATG11's chromosome transmission function (37°C enhances phenotypes)

• Genetic background effects:

  • Strain differences: S. cerevisiae strain backgrounds may have different baseline autophagy activity

  • Presence of tagged proteins: Tags may subtly alter protein function or interactions

• Methodology reconciliation:

  • Use multiple detection methods (Western blot, immunofluorescence, functional assays)

  • Perform side-by-side comparisons with standardized protocols

  • Consider kinetics: Some effects may be transient or time-dependent

When contradictions arise, it's important to determine whether they reflect true biological differences or technical artifacts. For example, research has shown that ATG11's role in chromosome transmission becomes more pronounced at elevated temperatures, which could lead to apparently contradictory results if temperature conditions vary between studies .

How might phospho-specific ATG11 antibodies advance our understanding of selective autophagy regulation?

Developing phospho-specific ATG11 antibodies would enable significant advances:

• Mapping phosphorylation dynamics: Track the temporal sequence of ATG11 phosphorylation events during autophagy initiation and progression.

• Stimulus-specific phosphorylation patterns: Distinguish between phosphorylation patterns induced by different stresses (nitrogen vs. glucose starvation vs. rapamycin) .

• Structure-function relationships: Correlate specific phosphorylation sites with ATG11's ability to bind different interaction partners .

• Signaling pathway integration: Determine how multiple kinases beyond ATG1 might regulate ATG11 through phosphorylation.

• Therapeutic target identification: Identify which phosphorylation events might serve as intervention points for modulating selective autophagy in disease contexts.

Research has established that ATG1-mediated phosphorylation of ATG11 is required for selective autophagy by regulating its association with receptor proteins . Phospho-specific antibodies would allow precise tracking of these regulatory events at the molecular level.

What innovative techniques might enhance ATG11 antibody applications in advanced microscopy?

Emerging techniques for ATG11 visualization include:

• Super-resolution microscopy approaches:

  • STORM/PALM imaging with specially conjugated ATG11 antibodies to visualize sub-diffraction structures

  • SIM (Structured Illumination Microscopy) to resolve ATG11 association with autophagic structures

• Live-cell imaging strategies:

  • Split-fluorescent protein complementation to visualize ATG11 interactions in real-time

  • FRAP (Fluorescence Recovery After Photobleaching) with fluorescently labeled antibody fragments to measure ATG11 dynamics

• Correlative light and electron microscopy (CLEM):

  • Use ATG11 antibodies conjugated to gold particles for precision localization at ultrastructural level

  • Combine with tomography to create 3D reconstructions of ATG11-containing structures

• Expansion microscopy:

  • Physical expansion of specimens to achieve super-resolution with standard confocal microscopy

  • Particularly useful for resolving ATG11's association with both autophagic structures and spindle components

• Proximity labeling approaches:

  • APEX2 or TurboID fusions to ATG11 to identify proximal proteins in different cellular compartments

  • Especially valuable for distinguishing autophagy-related versus chromosome transmission-related interaction networks

These advanced techniques would help resolve the spatial and temporal dynamics of ATG11's dual functions in autophagy and chromosome transmission with unprecedented detail.