ATG101 Antibody, HRP conjugated

What is ATG101 and what role does it play in cancer research?

ATG101 (Autophagy Related Gene 101) is a critical component of the autophagy pathway that plays a significant role in tumor development by responding to cellular stress. Research has shown that ATG101 expression is upregulated in multiple cancer types including breast cancer (BRCA), cholangiocarcinoma (CHOL), colorectal adenocarcinoma (COAD), esophageal carcinoma (ESCA), liver hepatocellular carcinoma (LIHC), and several others . ATG101 functions as part of the ULK1 complex and serves as a bridge between the ULK1 and PtdIns3K complexes, both essential for the initiation of autophagosome formation . Its C-terminal region has been identified as particularly important for this bridging function, making ATG101 a potential target and prognostic marker for tumor immunotherapy across different cancer types .

What are the advantages of using HRP-conjugated ATG101 antibodies in research?

Using HRP-conjugated ATG101 antibodies offers several significant advantages in research applications:

• Direct detection capability: The direct conjugation eliminates the need for secondary antibodies, reducing protocol complexity and potential cross-reactivity issues .

• Reduced background: Fewer antibody layers mean less non-specific binding, particularly valuable in tissues with high endogenous biotin or when using multiple antibodies .

• Time efficiency: HRP-conjugated primary antibodies streamline experimental protocols by eliminating secondary antibody incubation and washing steps, particularly valuable in time-consuming techniques .

• Enhanced sensitivity: Direct labeling can improve signal detection in samples with low ATG101 expression levels .

• Compatibility with multiple visualization systems: HRP can be detected using various substrates including diaminobenzidine (DAB), ABTS, TMB, and TMBUS, offering flexibility in experimental design .

What detection methods are most compatible with HRP-conjugated ATG101 antibodies?

HRP-conjugated ATG101 antibodies are compatible with multiple detection methodologies commonly used in molecular and cellular biology research:

• Western blotting: HRP-conjugated antibodies can be visualized using enhanced chemiluminescence (ECL) or chemifluorescence substrates to detect ATG101 protein expression levels .

• Immunohistochemistry (IHC): These antibodies can be applied to tissue sections where HRP activity is detected through chromogenic substrates like DAB, which produces a brown precipitate in the presence of hydrogen peroxide .

• Enzyme-linked immunosorbent assay (ELISA): HRP-conjugated ATG101 antibodies can quantify ATG101 in solution using colorimetric or fluorometric substrates .

• Immunocytochemistry (ICC): For cellular localization studies, HRP-conjugated antibodies allow visualization of ATG101 distribution, particularly valuable for studying its role in autophagosome formation .

• ChIP assays: With appropriate optimization, these antibodies can be used in chromatin immunoprecipitation experiments to investigate transcriptional regulation of ATG101 .

What buffer conditions are crucial when preparing HRP-conjugated ATG101 antibodies?

The composition of antibody buffers significantly affects the conjugation efficiency and stability of HRP-conjugated ATG101 antibodies. Key considerations include:

• Buffer additives: Common buffer additives can hamper the conjugation process. Researchers should verify that their antibody preparation is free from interfering components .

• pH optimization: Maintaining optimal pH during conjugation is essential as extreme pH can denature either the antibody or the HRP enzyme.

• Stability enhancers: Addition of stabilizing proteins like BSA (bovine serum albumin) at 0.1-0.5% can maintain antibody activity during long-term storage.

• Preservatives: Low concentrations of preservatives such as sodium azide (0.02-0.05%) can prevent microbial growth, though researchers should note that sodium azide can inhibit HRP activity at higher concentrations.

• Storage conditions: HRP-conjugated antibodies typically require storage at 2-8°C and should avoid repeated freeze-thaw cycles that can compromise the enzymatic activity of HRP.

The use of commercial conjugation kits like Lightning-Link® HRP can simplify this process and ensure optimal conjugation conditions .

What controls should be included when using HRP-conjugated ATG101 antibodies?

Proper experimental controls are essential for reliable results when using HRP-conjugated ATG101 antibodies:

• Negative controls:

  • Isotype control: An HRP-conjugated antibody of the same isotype but irrelevant specificity

  • No primary antibody control: Omitting the HRP-conjugated ATG101 antibody to assess background from detection reagents

  • Tissues/cells known to be negative for ATG101 expression

• Positive controls:

  • Tissues/cells with confirmed ATG101 expression (particularly breast cancer, cholangiocarcinoma, and liver hepatocellular carcinoma tissues)

  • Recombinant ATG101 protein standards for quantitative applications

• Specificity controls:

  • Pre-absorption control: Pre-incubating the HRP-conjugated ATG101 antibody with purified ATG101 protein

  • siRNA/shRNA knockdown of ATG101 in experimental samples

• Technical controls:

  • Loading controls for Western blots (β-actin, GAPDH)

  • Internal staining controls for IHC/ICC (tissues with consistent ATG101 expression levels)

How should I optimize the protocol for Western blotting with HRP-conjugated ATG101 antibodies?

Optimizing Western blotting protocols for HRP-conjugated ATG101 antibodies requires attention to several key parameters:

• Sample preparation:

  • Use fresh tissue/cell lysates with protease inhibitors to prevent ATG101 degradation

  • Ensure equal protein loading (20-50 μg total protein per lane)

  • Include phosphatase inhibitors if studying ATG101 phosphorylation states

• Electrophoresis conditions:

  • Use 10-12% SDS-PAGE gels for optimal resolution of ATG101 (approximately 25 kDa)

  • Include molecular weight markers covering the 15-35 kDa range

• Transfer parameters:

  • Semi-dry or wet transfer at 100-120V for 60-90 minutes (or 30V overnight at 4°C)

  • Use PVDF membranes for highest protein binding capacity and signal intensity

• Blocking conditions:

  • 5% non-fat dry milk or 3-5% BSA in TBST for 1 hour at room temperature

  • Optimize blocking time to minimize background without affecting specific signal

• Antibody dilution:

  • Typically 1:1000 to 1:5000 dilution in blocking buffer (optimize through titration)

  • Incubate for 2 hours at room temperature or overnight at 4°C

• Washing steps:

  • 3-5 washes with TBST, 5-10 minutes each

  • Thorough washing is particularly important with direct HRP-conjugated antibodies

• Detection:

  • Use fresh ECL substrate with appropriate exposure times

  • Start with short exposures (30 seconds) and increase as needed to avoid overexposure

How can HRP-conjugated ATG101 antibodies be used to investigate the interaction between ATG101 and autophagy complexes?

HRP-conjugated ATG101 antibodies provide valuable tools for investigating the critical interactions between ATG101 and autophagy-related complexes:

• Co-immunoprecipitation assays:

  • HRP-conjugated ATG101 antibodies can be used to pull down ATG101 and its binding partners, particularly components of the ULK1 and PtdIns3K complexes

  • The C-terminal region of ATG101, which bridges ULK1 and PtdIns3K complexes, can be specifically studied through targeted co-IP experiments

• Proximity ligation assays (PLA):

  • These antibodies can be adapted for PLA to visualize and quantify interactions between ATG101 and other autophagy proteins in situ

  • This approach is particularly valuable for studying the dynamics of complex formation during autophagy induction

• Chromatin immunoprecipitation (ChIP) assays:

  • HRP-conjugated ATG101 antibodies can be utilized in ChIP experiments to identify DNA binding sites and transcription factors associated with ATG101

  • Primer sequences for the amplification of human ATG101 promoter associated with transcription factors like EGR2 have been validated (5′-CTGGTCGTGGACTGTGGTTG-3′ forward and 5′-CGGAAGCTGGAGGAGCG-3′ reverse)

• Structure-function studies:

  • HRP-conjugated antibodies targeting specific domains of ATG101 can help elucidate the functional significance of different regions

  • Particularly valuable for studying the C-terminal segment which adopts different conformations (β-strand in free ATG101 versus α-helix or random coil when complexed with ATG13)

What approaches can be used to correlate ATG101 expression with immune checkpoint activity in cancer?

Recent research highlights important connections between ATG101 expression and immune checkpoint activity in cancer, which can be investigated using HRP-conjugated ATG101 antibodies:

• Multiplex immunohistochemistry:

  • Combining HRP-conjugated ATG101 antibodies with differently labeled antibodies against immune checkpoint molecules (PD-L1, CTLA-4)

  • This approach requires careful optimization of antigen retrieval and detection systems to prevent cross-reactivity

• Correlative tissue analysis:

  • Serial tissue sections can be stained for ATG101 and immune checkpoint molecules to assess their co-expression patterns

  • Spearman correlation tests can be used to analyze the relationship between ATG101 expression and various immune checkpoint target genes (up to 47 have been studied)

• Functional assays:

  • In vitro systems using HRP-conjugated ATG101 antibodies can examine how modulation of ATG101 expression affects immune checkpoint activity

  • Particular attention should be paid to the relationship between ATG101 and PD-L1, given emerging bispecific therapies like ATG-101

• Tumor microenvironment analysis:

  • HRP-conjugated ATG101 antibodies can be used to correlate ATG101 expression with immune cell infiltration

  • This approach is valuable for understanding how ATG101 impacts CD8+ T cells, regulatory T cells, and effector memory T cells in the tumor microenvironment

Table 1: Correlation between ATG101 expression and immune checkpoint molecules in selected cancer types

Note: Correlation coefficients (r) are representative and should be validated in specific experimental contexts.

How can ATG101 and methylation status be studied using HRP-conjugated antibodies?

DNA methylation plays a crucial role in tumor development, and the relationship between ATG101 and methylation status can be studied using HRP-conjugated antibodies:

• Combined methylation and expression analysis:

  • HRP-conjugated ATG101 antibodies can be used to assess protein expression in parallel with DNA methylation analysis

  • This approach can determine if ATG101 expression correlates with methylation of its own promoter or other cancer-related genes

• Investigation of methyltransferase relationships:

  • Research has shown correlations between ATG101 expression and four methyltransferases (DNMT1, DNMT2, DNMT3A and DNMT3B)

  • HRP-conjugated ATG101 antibodies can be used in co-immunoprecipitation assays to investigate physical interactions with these methyltransferases

• Chromatin immunoprecipitation followed by bisulfite sequencing:

  • This approach can determine if ATG101 is associated with differentially methylated regions of chromatin

  • HRP-conjugated ATG101 antibodies can facilitate the immunoprecipitation step of this technique

• Analysis of methylation inhibitors:

  • HRP-conjugated ATG101 antibodies can assess how treatment with DNA methylation inhibitors affects ATG101 expression

  • This approach helps elucidate epigenetic regulation of ATG101 in cancer contexts

How can I address non-specific binding when using HRP-conjugated ATG101 antibodies?

Non-specific binding is a common challenge when using HRP-conjugated antibodies. For ATG101 detection, consider these approaches:

• Optimization of blocking conditions:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time to 2 hours at room temperature

  • Add 0.1-0.3% Triton X-100 or 0.05% Tween-20 to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Perform a titration series (1:500 to 1:5000) to determine optimal antibody concentration

  • Use the highest dilution that gives specific signal to minimize background

• Sample preparation improvements:

  • Include additional washing steps with higher salt concentration (up to 500 mM NaCl)

  • Pre-absorb the antibody with proteins from the species being tested

  • Treat samples with commercial background reducers before antibody application

• Detection system modifications:

  • Use substrate with lower sensitivity for high-expression systems

  • Shorten substrate incubation time to minimize background development

  • Consider alternative detection methods if HRP is causing persistent background

How should I interpret discrepancies between ATG101 protein levels and autophagy markers?

Interpreting discrepancies between ATG101 expression and other autophagy markers requires careful consideration of several factors:

• Temporal dynamics of autophagy:

  • ATG101 functions early in autophagosome formation, while other markers (LC3-II, p62) reflect different stages

  • Consider examining multiple time points to capture the complete autophagic flux

• Context-dependent regulation:

  • In cancer cells, alternative pathways may compensate for alterations in ATG101 expression

  • The relationship between ATG101 and other autophagy components may be tissue-specific

• Post-translational modifications:

  • ATG101 function may be regulated by phosphorylation or other modifications not reflected by total protein levels

  • Consider using phospho-specific antibodies alongside total ATG101 detection

• Interpretation framework:

  • When ATG101 is high but autophagy appears low: Consider inhibition at downstream steps or alternative roles of ATG101

  • When ATG101 is low but autophagy appears high: Evaluate potential compensatory mechanisms or alternative autophagy pathways

Table 2: Interpretation guide for ATG101 and autophagy marker patterns

What statistical approaches are recommended for analyzing ATG101 expression in relation to patient prognosis?

When analyzing the relationship between ATG101 expression and patient outcomes, several statistical approaches are recommended:

How might HRP-conjugated ATG101 antibodies contribute to research on bispecific antibody therapies?

HRP-conjugated ATG101 antibodies could play a valuable role in research related to bispecific antibody therapies like ATG-101 (PD-L1×4-1BB):

• Target expression profiling:

  • HRP-conjugated ATG101 antibodies can help characterize ATG101 expression in tumors being considered for bispecific antibody treatment

  • This may identify additional cancer types that could benefit from immunotherapeutic approaches

• Mechanistic studies:

  • These antibodies can help elucidate how autophagy pathways (involving ATG101) interact with immune checkpoint mechanisms

  • Understanding this crosstalk is crucial as bispecific antibodies like ATG-101 target immune checkpoint molecules such as PD-L1

• Resistance mechanism investigation:

  • HRP-conjugated ATG101 antibodies can examine how autophagy contributes to resistance against immune checkpoint inhibitors

  • This research is particularly relevant as ATG-101 is designed to address resistance to conventional immune checkpoint inhibitors

• Biomarker development:

  • ATG101 expression levels, detected with HRP-conjugated antibodies, could potentially serve as biomarkers for response to bispecific therapies

  • Correlative studies could determine if baseline ATG101 expression predicts response to therapies like ATG-101

What novel methodologies might enhance the utility of HRP-conjugated ATG101 antibodies?

Several emerging methodologies could enhance the research applications of HRP-conjugated ATG101 antibodies:

• Spatial transcriptomics integration:

  • Combining HRP-based protein detection with spatial transcriptomics to correlate ATG101 protein expression with local transcriptional signatures

  • This approach could reveal microenvironmental factors influencing ATG101 expression in tumors

• Advanced multiplexing techniques:

  • Sequential HRP labeling and quenching to perform highly multiplexed imaging with multiple antibodies including ATG101

  • This would allow simultaneous visualization of ATG101 with numerous autophagy and immune markers

• Three-dimensional tissue analysis:

  • Adapting HRP-conjugated ATG101 antibodies for tissue clearing and light-sheet microscopy

  • This would enable whole-organ mapping of ATG101 expression patterns in experimental models

• Single-cell proteomics:

  • Utilizing HRP-conjugated ATG101 antibodies in mass cytometry or single-cell proteomics workflows

  • This could identify rare cell populations with unique ATG101 expression patterns within heterogeneous tumors

• Functional CRISPR screening:

  • Using HRP-conjugated ATG101 antibodies as readouts in CRISPR-based functional genomics screens

  • This approach could identify genes that regulate ATG101 expression or function in autophagy and cancer contexts