ATG101 Antibody

What is ATG101 and what is its role in autophagy?

ATG101 (Autophagy-related protein 101) is a crucial factor required for autophagosome formation in the autophagy pathway. It functions primarily by stabilizing ATG13, protecting it from proteasomal degradation . The protein is also known by alternative names including C12orf44 and PP894 . Autophagy, the process of bulk degradation of cellular proteins through an autophagosomic-lysosomal pathway, is important for normal growth control and may be defective in tumor cells . ATG101 is part of the ULK1 complex that initiates autophagy, particularly under nutrient starvation conditions, making it an important target for researchers studying cellular stress responses, cancer biology, and neurodegenerative diseases where autophagy plays a significant role.

What applications are ATG101 antibodies suitable for?

Based on validated research applications, ATG101 antibodies are suitable for several experimental techniques:

When selecting an application, researchers should consider that some antibodies may perform better in specific applications than others, and optimization for your specific experimental conditions is recommended .

What is the expected molecular weight of ATG101 in Western blot applications?

While the calculated molecular weight of ATG101 is approximately 25 kDa (from its 218 amino acid sequence) , researchers should be aware that observed molecular weights can vary. Western blot analyses have shown:

• 25-28 kDa bands in human liver tissue lysate and lung carcinoma cell lines

• A higher 68 kDa band has been reported in some applications, potentially representing a post-translationally modified form or complex of the protein

This variability underscores the importance of including appropriate positive controls in Western blot experiments to confirm antibody specificity and expected band sizes in your specific experimental system.

How should ATG101 antibodies be stored to maintain reactivity?

For optimal preservation of antibody activity, ATG101 antibodies should be stored as follows:

• Short-term storage (up to three months): 4°C

• Long-term storage (up to one year): -20°C

It is crucial to avoid repeated freeze-thaw cycles as these can degrade antibody quality and reduce specificity and sensitivity . Additionally, antibodies should not be exposed to prolonged high temperatures. Most commercial ATG101 antibodies are supplied in PBS containing preservatives such as sodium azide (0.02%) , which helps maintain stability during storage.

How can I validate ATG101 antibody specificity in my experimental system?

Validating antibody specificity is critical for reliable results. For ATG101 antibodies, consider implementing these validation approaches:

• Positive and negative control tissues/cell lines:

  • Validated positive controls include human lung carcinoma cell lines (NCI-H1299, A549) , human liver tissue , and mouse brain tissue

  • Use tissues/cells with known ATG101 knockout or knockdown as negative controls

• Molecular weight verification:

  • Confirm band presence at the expected 25-28 kDa range

  • Be aware that observed molecular weights may vary from the calculated 25 kDa due to post-translational modifications

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide (available for purchase from some manufacturers)

  • Loss of signal indicates specificity for the target epitope

• Orthogonal validation:

  • Compare results using antibodies targeting different epitopes of ATG101

  • Correlate protein detection with mRNA expression data

• Signal reduction after target knockdown:

  • Verify decreased signal intensity in ATG101 siRNA or shRNA-treated samples

These validation steps are particularly important when studying ATG101 in novel experimental systems or when publishing results in high-impact journals.

What are the considerations for using ATG101 antibodies in cancer research?

ATG101 has emerging significance in cancer research, with several important considerations:

• Expression patterns:

  • ATG101 expression has been studied in lung carcinoma cell lines (A549, NCI-H1299)

  • Recent research has identified ATG101 as a potential immune-related biomarker in cholangiocarcinoma cells after photodynamic therapy

• Relation to immunotherapy resistance:

  • While not directly about the ATG101 protein, research on the bispecific antibody ATG-101 (targeting PD-L1 and 4-1BB) has shown promise in treating tumors resistant to immune checkpoint inhibitors

  • This suggests potential interplay between autophagy pathways and immune checkpoint resistance mechanisms

• Methodological considerations:

  • For IHC applications in cancer tissues, antigen retrieval with TE buffer (pH 9.0) or alternatively citrate buffer (pH 6.0) is recommended

  • For xenograft models, antibody dilutions of approximately 1:500 have been validated

• Autophagy modulation in cancer:

  • When studying ATG101 in cancer contexts, consider its role in both pro-survival and pro-death autophagy mechanisms

  • Correlate ATG101 expression with other autophagy markers to provide context for its activity

Understanding ATG101's role within the broader autophagy network is essential when evaluating its significance in different cancer types and therapeutic contexts.

How can I differentiate between ATG101 protein and the bispecific antibody ATG-101 in literature and research?

This is a critical distinction in the current literature:

• ATG101 (protein):

  • An endogenous human protein (25 kDa) involved in autophagy

  • Also known as C12orf44 or PP894

  • The target protein detected by anti-ATG101 antibodies

• ATG-101 (therapeutic antibody):

  • A tetravalent "2+2" PD-L1×4-1BB bispecific antibody engineered for cancer immunotherapy

  • Binds both PD-L1 and 4-1BB concurrently, with greater affinity for PD-L1

  • Activates 4-1BB+ T cells when cross-linked with PD-L1–positive cells

  • Demonstrates antitumor activity in tumor models resistant to immune checkpoint inhibitors

When reviewing literature, carefully examine context clues:

• Papers discussing autophagy mechanisms likely refer to the ATG101 protein

• Publications about cancer immunotherapy, particularly those mentioning PD-L1 and 4-1BB, likely refer to the bispecific antibody ATG-101

• Note the formatting: ATG101 (no hyphen) typically refers to the protein, while ATG-101 (with hyphen) refers to the bispecific antibody

This distinction is particularly important when conducting literature searches and evaluating research relevance to your specific aims.

What are the optimal conditions for immunohistochemistry (IHC) using ATG101 antibodies?

For successful IHC with ATG101 antibodies, the following protocol optimizations are recommended:

• Fixation and tissue preparation:

  • Formalin-fixed paraffin-embedded (FFPE) tissues have been validated

  • Human liver cancer tissue and xenograft tissues (e.g., A549 lung carcinoma) have been successfully used

• Antigen retrieval:

  • Primary recommendation: TE buffer at pH 9.0

  • Alternative method: Citrate buffer at pH 6.0

  • Complete antigen retrieval is critical for accessing the epitope, which may be masked during fixation

• Antibody dilution range:

  • Starting dilution: 1:50-1:500

  • For paraffin-embedded A549 xenograft tissue: 1:500 dilution has been validated

  • Titration is recommended for each new tissue type or experimental system

• Detection systems:

  • Both chromogenic (DAB) and fluorescent secondary detection systems are compatible

  • When using fluorescent detection, consider tissue autofluorescence, particularly in liver samples

• Controls:

  • Positive control: Human liver cancer tissue

  • Negative control: Omit primary antibody or use isotype control

Optimization of these conditions for your specific tissue of interest is essential for generating reproducible and meaningful IHC results.

What are the best practices for quantifying ATG101 in Western blot analysis?

For accurate quantification of ATG101 by Western blot, implement these methodological considerations:

• Sample preparation:

  • Validated lysates include NCI-H1299 whole cell extract (30 μg loading) and human liver tissue lysate (15 μg loading)

  • Use complete protease inhibitor cocktails during extraction to prevent degradation

  • Consider phosphatase inhibitors if studying post-translational modifications

• Gel selection:

  • 12% SDS-PAGE gels have been successfully used for resolving ATG101

  • Given the 25 kDa size, avoid gels with too low acrylamide percentage

• Antibody concentrations:

  • Primary antibody dilutions: 1:500-1:1000 (ab229235) , 1-2 μg/mL (ab105387) , or 1:500-1:800 (Proteintech)

  • Optimize blocking conditions to reduce background (typically 5% non-fat milk or BSA)

• Normalization controls:

  • Always include loading controls (β-actin, GAPDH, or total protein stains)

  • For comparative studies, consider normalizing to multiple housekeeping proteins

• Quantification approach:

  • Use digital imaging and densitometry software for quantification

  • Ensure signal is in the linear range of detection

  • For studies of autophagy flux, correlate ATG101 levels with other autophagy markers (LC3-II, p62)

• Statistical analysis:

  • Perform at least three biological replicates for quantitative comparisons

  • Use appropriate statistical tests based on data distribution

Following these practices will yield more reproducible and publishable quantitative Western blot data for ATG101.

How should I design experiments to study ATG101's role in the autophagy pathway?

To effectively investigate ATG101's functional role in autophagy, consider this experimental framework:

• Expression manipulation strategies:

  • siRNA/shRNA knockdown of ATG101 to assess loss-of-function effects

  • CRISPR/Cas9 knockout for complete elimination of ATG101

  • Overexpression studies using tagged constructs (consider epitope tags that won't interfere with ATG101-ATG13 interaction)

• Functional autophagy assays:

  • LC3 puncta formation (by fluorescence microscopy)

  • LC3-I to LC3-II conversion (by Western blot)

  • p62/SQSTM1 degradation

  • Autophagic flux measurement using lysosomal inhibitors (bafilomycin A1, chloroquine)

  • Long-lived protein degradation assays

• Interaction studies:

  • Co-immunoprecipitation to confirm ATG101-ATG13 interaction

  • Proximity ligation assays to visualize interactions in situ

  • Assess effects on ULK1 complex formation and stability

• Subcellular localization:

  • Track ATG101 localization under various conditions (nutrient starvation, mTOR inhibition)

  • Co-localization with other autophagy markers

• Context-dependent regulation:

  • Examine ATG101 expression and function under different stress conditions:

    • Nutrient starvation (EBSS media)

    • mTOR inhibition (rapamycin, Torin1)

    • ER stress (tunicamycin, thapsigargin)

    • Hypoxia

• Single-cell analysis:

  • Consider single-cell RNA sequencing to assess heterogeneity in ATG101 expression and function

  • This approach has been valuable in characterizing immune landscapes in tumor microenvironments

This multifaceted experimental approach will provide comprehensive insights into ATG101's specific contributions to autophagy regulation in your biological system of interest.

What are common troubleshooting strategies for weak or absent ATG101 signal in Western blots?

When encountering weak or absent ATG101 signal in Western blot experiments, consider these systematic troubleshooting approaches:

• Sample preparation issues:

  • Ensure complete cell lysis (consider RIPA buffer for more stringent extraction)

  • Check protein concentration measurement accuracy

  • Increase loading amount (validated protocols use 15-30 μg of total protein)

  • Add fresh protease inhibitors to prevent degradation

• Antibody-specific considerations:

  • Verify antibody expiration date and storage conditions

  • Increase antibody concentration (try 1:500 instead of 1:1000, or 2 μg/mL instead of 1 μg/mL)

  • Extended primary antibody incubation (overnight at 4°C)

  • Test alternative ATG101 antibodies targeting different epitopes

• Detection system optimization:

  • Use more sensitive detection methods (enhanced chemiluminescence plus or fluorescent secondary antibodies)

  • Increase exposure time incrementally

  • Check secondary antibody compatibility and freshness

• Technical parameters:

  • Optimize transfer conditions (time, current, buffer composition)

  • Ensure appropriate blocking (5% milk may be too stringent; try 3% BSA)

  • Reduce washing stringency (lower salt concentration or fewer washes)

• Biological considerations:

  • Verify ATG101 expression in your cell type/tissue (check RNA-seq databases)

  • Consider induction of autophagy to upregulate ATG101 (starvation, rapamycin)

  • Some cell lines may have naturally low ATG101 expression

• Positive controls:

  • Include validated positive controls: human lung carcinoma cell lines, human liver tissue, or mouse brain tissue

Systematic evaluation of these factors will help identify and resolve the source of weak ATG101 signal in Western blot applications.

How can I address cross-reactivity or non-specific binding when using ATG101 antibodies?

To minimize cross-reactivity and non-specific binding with ATG101 antibodies:

• Antibody selection:

  • Choose antibodies validated for your specific application and species

  • Polyclonal antibodies (like those in the search results) may have higher cross-reactivity risk than monoclonals

  • Review validation data from manufacturers showing single bands at expected molecular weight

• Blocking optimization:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Increase blocking time or concentration for high-background samples

  • Add 0.1-0.3% Tween-20 to reduce non-specific hydrophobic interactions

• Antibody dilution optimization:

  • Titrate antibody concentrations to find optimal signal-to-noise ratio

  • Higher dilutions (e.g., 1:1000 vs 1:500) may reduce non-specific binding

• Washing stringency:

  • Increase number of washes or duration

  • Use TBS-T with higher Tween-20 concentration for more stringent washing

• Absorption controls:

  • Pre-absorb antibody with the immunizing peptide where available

  • This can confirm which bands are specific vs. non-specific

• Species-specific considerations:

  • When working with tissue samples, use species-specific secondary antibodies

  • Consider species cross-reactivity when working with mixed species samples

• Validation approaches:

  • Test antibody in ATG101 knockdown/knockout samples

  • Compare staining patterns across multiple antibodies targeting different ATG101 epitopes

These strategies should significantly reduce non-specific binding issues with ATG101 antibodies across different experimental applications.

What quality control measures should be implemented when using ATG101 antibodies for publication-quality research?

For publication-quality research using ATG101 antibodies, implement these rigorous quality control measures:

• Antibody validation documentation:

  • Document complete antibody information (manufacturer, catalog number, lot number, RRID)

  • Include validation of antibody specificity in your experimental system

  • Consider using antibodies cited in peer-reviewed publications

• Experimental controls:

  • Positive controls: Include samples known to express ATG101

  • Negative controls: Include antibody omission controls and ideally ATG101 knockdown/knockout samples

  • Isotype controls: Particularly important for IHC/IF applications

• Technical replication:

  • Perform at least three independent biological replicates

  • Include technical replicates within each experiment

  • Document consistency across replicates with appropriate statistical analysis

• Quantification standards:

  • Use digital image capture with consistent exposure settings

  • Apply standardized quantification methods (region selection, background subtraction)

  • Include calibration standards when appropriate

• Multi-method confirmation:

  • Verify key findings with orthogonal methods (e.g., IF and WB)

  • Correlate protein detection with mRNA expression data

  • Consider mass spectrometry validation for critical findings

• Data transparency:

  • Show full blots/gels with molecular weight markers visible

  • Indicate any image adjustments applied (contrast, brightness)

  • Maintain raw, unprocessed image files for potential journal requests

• Method documentation:

  • Provide detailed methods including blocking conditions, antibody dilutions, incubation times and temperatures

  • Document antigen retrieval methods for IHC (TE buffer pH 9.0 or citrate buffer pH 6.0)

Following these quality control measures will strengthen the reliability and reproducibility of research findings involving ATG101 antibodies, increasing the likelihood of acceptance in high-impact journals.