Phospho-ATR (Ser428) Antibody

What is ATR and why is phosphorylation at Ser428 significant?

ATR (Ataxia Telangiectasia and Rad3-related protein) is a ~300 kDa serine/threonine kinase that plays a critical role in DNA damage response and cell cycle checkpoint activation. ATR is activated in response to various forms of DNA damage, particularly single-stranded DNA breaks that can arise from replication stress or UV radiation. Phosphorylation at Serine 428 represents one of the key post-translational modifications associated with ATR activation. This phosphorylation event is considered a biomarker for active ATR signaling in the DNA damage response pathway, making antibodies against this phospho-site valuable tools for investigating cellular responses to genotoxic stress .

What are the key specifications of commercially available Phospho-ATR (Ser428) antibodies?

Phospho-ATR (Ser428) antibodies are typically rabbit polyclonal antibodies that specifically recognize ATR when phosphorylated at Serine 428. These antibodies have the following general specifications:

The antibodies are typically generated using synthetic phosphopeptides corresponding to the region surrounding Serine 428 of human ATR protein .

How are Phospho-ATR (Ser428) antibodies validated?

These antibodies are commonly validated through multiple approaches:

• Western blotting using positive controls such as UV-irradiated cell lysates (HeLa, COS1), which triggers ATR phosphorylation

• Peptide competition assays comparing binding with phospho-peptides versus non-phospho peptides

• Cross-reactivity testing across multiple species

• Specificity assessment in various cell types with known ATR expression levels

Validation typically demonstrates an approximately 260-300 kDa band corresponding to phosphorylated ATR that can be competed away using the specific phosphopeptide but not with non-phosphorylated peptide .

What is the recommended protocol for Western blotting with Phospho-ATR (Ser428) antibody?

Sample Preparation:

• Collect cells at exponential growth phase

• If studying DNA damage response, treat cells with DNA damaging agents (e.g., UV radiation, hydroxyurea)

• Wash cells with cold PBS

• Lyse cells in a buffer containing phosphatase inhibitors to preserve phosphorylation status

• Centrifuge lysate and collect supernatant

• Quantify protein concentration

Western Blotting Protocol:

• Load 10-20 μg of lysate per lane on 6-8% SDS-PAGE gels (optimized for high molecular weight proteins)

• Transfer proteins to PVDF membrane (extended transfer time recommended for large proteins)

• Block membrane with 5% BSA in TBST (not milk, as it contains phosphatases)

• Incubate with Phospho-ATR (Ser428) antibody at 1:1000 dilution overnight at 4°C

• Wash membrane 3-5 times with TBST

• Incubate with HRP-conjugated secondary antibody

• Develop using enhanced chemiluminescence detection system

Critical Considerations:

• Due to the high molecular weight of ATR (300 kDa), use low percentage gels and extend transfer time

• Always include phosphatase inhibitors throughout sample preparation

• Consider using positive controls (UV-irradiated lysates) and negative controls

How can I optimize immunohistochemistry (IHC) protocols for Phospho-ATR (Ser428) detection?

For antibodies supporting IHC applications , the following optimization steps are recommended:

• Tissue Preparation:

  • Use freshly prepared 4% paraformaldehyde-fixed, paraffin-embedded sections

  • Consider testing both heat-induced and enzymatic antigen retrieval methods

• Staining Protocol:

  • Deparaffinize and rehydrate sections

  • Perform antigen retrieval (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Block endogenous peroxidase with 3% H₂O₂

  • Apply protein block (5% normal goat serum)

  • Incubate with primary antibody at 1:100-1:200 dilution (optimize for your tissue)

  • Use appropriate detection system (HRP-polymer or biotin-streptavidin)

  • Counterstain, dehydrate, and mount

• Controls and Validation:

  • Include positive control tissues with known ATR activation

  • Use phosphatase-treated serial sections as negative controls

  • Consider dual staining with total ATR antibody to confirm specificity

What stimuli can be used to induce ATR phosphorylation for positive controls?

Several treatments can reliably induce ATR phosphorylation at Ser428:

• UV irradiation (10-50 J/m²) with 1-2 hour recovery time

• Hydroxyurea (2-5 mM for 4-24 hours)

• Aphidicolin (1-5 μg/ml for 12-24 hours)

• Camptothecin (1-5 μM for 2-4 hours)

• Cisplatin (10-50 μM for 12-24 hours)

UV irradiation is often considered the gold standard for ATR activation and is commonly used in validation studies . The most effective treatment may vary by cell type, so optimization is recommended when establishing a new experimental system.

Why might I fail to detect Phospho-ATR (Ser428) in my Western blot?

Several technical factors can affect Phospho-ATR (Ser428) detection:

• Protein Degradation: ATR is a large protein susceptible to degradation. Ensure complete protease inhibitor cocktails are used during sample preparation.

• Loss of Phosphorylation: Phosphorylation can be lost during sample preparation. Always include phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) in lysis buffers.

• Insufficient Activation: The stimulus may not have effectively activated ATR. Verify activation using known stimuli like UV irradiation as positive controls.

• Inefficient Transfer: Large proteins like ATR (300 kDa) transfer inefficiently. Use lower percentage gels (6-8%), extend transfer time, or consider wet transfer methods.

• Antibody Specificity Issues: Confirm the antibody recognizes your species of interest. Some Phospho-ATR (Ser428) antibodies have limited cross-reactivity .

• Signal Detection Limitations: ATR may be expressed at low levels. Consider using enhanced chemiluminescence substrates or increase protein loading.

How can I distinguish between specific and non-specific bands when using Phospho-ATR (Ser428) antibody?

To distinguish between specific and non-specific signals:

• Molecular Weight Verification: Phospho-ATR should appear at approximately 300 kDa. Bands at other molecular weights may represent non-specific binding or degradation products.

• Peptide Competition: Perform peptide competition assays using the phospho-peptide immunogen. The specific band should disappear when the antibody is pre-incubated with the phosphopeptide but remain with the non-phosphopeptide .

• Validation with Multiple Antibodies: Confirm results using different Phospho-ATR (Ser428) antibody clones or antibodies against other ATR phosphorylation sites.

• Knockdown/Knockout Controls: Use ATR siRNA knockdown or CRISPR knockout cells as negative controls.

• Activation/Inhibition Treatments: The signal should increase with ATR-activating treatments (UV, replication stress) and decrease with ATR kinase inhibitors.

What are the storage and handling recommendations for maintaining antibody performance?

To maintain optimal antibody performance:

• Storage Temperature: Store antibodies at 2-8°C for short-term (1 year) or aliquot and store at -20°C for long-term storage .

• Avoid Freeze-Thaw Cycles: Repeated freeze-thaw cycles can degrade antibodies. Prepare small aliquots before freezing.

• Working Dilution Storage: Diluted antibody can typically be stored at 4°C for up to one week. For longer storage, add preservatives like sodium azide (0.05%).

• Contamination Prevention: Use sterile technique when handling antibodies to prevent microbial contamination.

• Centrifugation Before Use: Briefly centrifuge antibody vials before opening to collect liquid at the bottom and avoid material in the cap.

How can Phospho-ATR (Ser428) antibodies be used to investigate the DNA damage response pathway?

Phospho-ATR (Ser428) antibodies can be employed in several advanced applications:

• Kinetics of ATR Activation: Monitor the temporal dynamics of ATR phosphorylation following DNA damage by performing time-course experiments with various genotoxic agents.

• Pathway Analysis: Use Phospho-ATR (Ser428) antibodies in combination with antibodies against downstream targets (Chk1, p53, H2AX) to characterize the complete signaling cascade.

• Drug Response Studies: Evaluate how novel chemotherapeutic compounds affect ATR phosphorylation status as a biomarker of DNA damage response activation.

• Synthetic Lethality Screening: Identify genes or compounds that, when combined with ATR inhibition, produce synergistic cell death in cancer cells.

• Cell Cycle Analysis: Combine with cell cycle markers to determine when during the cell cycle ATR becomes phosphorylated in response to specific stressors.

Research has demonstrated that ATR phosphorylation is crucial for mediating responses to replication stress and maintaining genomic stability, making it a valuable biomarker for research in cancer biology and DNA damage response mechanisms .

What are recommended controls for Phospho-ATR (Ser428) experiments in different experimental contexts?

For robust experimental design, incorporate these controls:

For Western Blotting:

• Positive Control: UV-irradiated HeLa or COS1 cell lysates

• Negative Control: ATR inhibitor-treated cells or ATR knockdown/knockout cells

• Phosphatase Control: Lysate treated with lambda phosphatase to demonstrate phospho-specificity

• Loading Control: Antibody against housekeeping protein or total ATR

For Immunohistochemistry:

• Positive Control Tissue: Tissues with known ATR activation (e.g., certain tumors)

• Negative Control Staining: Primary antibody omission or isotype control

• Phosphatase-Treated Control: Serial section treated with phosphatase

• Antibody Validation Control: Peptide competition

For Drug Studies:

• Vehicle Control: Cells treated with solvent alone

• Time Course Control: Multiple time points to capture activation kinetics

• Dose Response Control: Multiple concentrations to determine sensitivity

• Pathway Validation: Parallel assessment of known ATR substrates (e.g., phospho-Chk1)

How can I use Phospho-ATR (Ser428) antibodies for multiplexed analysis of DNA damage response pathways?

Advanced multiplexed applications include:

• Multi-Color Immunofluorescence:

  • Co-stain for Phospho-ATR (Ser428) with other DDR markers (γH2AX, 53BP1, Rad51)

  • Use spectrally distinct fluorophores for simultaneous detection

  • Perform quantitative image analysis to correlate different markers at the single-cell level

• Flow Cytometry Application:

  • Combine with cell cycle markers (propidium iodide, DAPI) and other DDR proteins

  • Analyze correlations between ATR activation and cell cycle phase

  • Evaluate heterogeneity in cellular responses to DNA damage

• Proximity Ligation Assay (PLA):

  • Detect interactions between phosphorylated ATR and partner proteins

  • Visualize active ATR complexes at sites of DNA damage

  • Quantify interactions in different subcellular compartments

• Mass Cytometry (CyTOF):

  • Label ATR with metal-conjugated antibodies

  • Perform high-dimensional analysis with 30+ DDR markers simultaneously

  • Create comprehensive profiles of DDR pathway activation

• Sequential Immunoblotting:

  • Strip and reprobe membranes to detect multiple phospho-proteins

  • Create comprehensive signaling profiles from limited samples

  • Establish activation hierarchies within signaling cascades

How should I quantify and normalize Phospho-ATR (Ser428) Western blot signals?

For accurate quantification:

• Capture Optimal Images: Ensure signals are within the linear dynamic range of your detection system, avoiding saturation.

• Normalization Approaches:

  • Normalize to total ATR protein (ideal but requires stripping and reprobing)

  • Normalize to loading controls (GAPDH, β-actin), though these may not be ideal due to the size difference

  • Consider normalizing to total protein using stain-free gels or Ponceau S staining

• Quantification Method:

  • Use densitometry software (ImageJ, Image Lab, etc.)

  • Define consistent region of interest for all bands

  • Subtract background from an adjacent area

  • Calculate relative intensity compared to control samples

• Statistical Analysis:

  • Perform experiments in biological triplicates minimum

  • Apply appropriate statistical tests based on data distribution

  • Present data as fold change relative to control conditions

• Presentation Format:

  • Show representative blots with molecular weight markers

  • Include quantification graphs with error bars

  • Clearly state normalization method in figure legends

What are the potential pitfalls when interpreting Phospho-ATR (Ser428) results in the context of DNA damage responses?

Several considerations may affect data interpretation:

• Temporal Dynamics: ATR phosphorylation is dynamic and time-dependent. A negative result at a single time point may miss the activation window.

• Cell Type Variations: Different cell types show varying baseline levels and induction kinetics of ATR phosphorylation. Direct comparisons between cell types should be made cautiously.

• Protein Expression Levels: Overexpression systems may show different phosphorylation patterns than endogenous proteins. Confirm key findings in systems with physiological expression levels.

• Indirect Activation: Some stimuli may activate ATR indirectly through other pathways. Use pathway inhibitors to confirm the direct relationship between stimulus and ATR phosphorylation.

• Partial Activation: Phosphorylation at Ser428 represents only one aspect of ATR activation. Consider examining multiple phosphorylation sites and downstream targets for a complete picture.

• Threshold Effects: ATR signaling may exhibit threshold effects where small changes in phosphorylation translate to significant biological outcomes. Quantitative analysis is essential.

• Complex Formation: ATR functions in complexes with other proteins (e.g., ATRIP). Phosphorylation may not directly correlate with functional activity without considering complex formation.

How can Phospho-ATR (Ser428) data be integrated with other biomarkers to build a comprehensive DNA damage response profile?

To develop comprehensive DDR profiles:

• Multi-marker Analysis: Combine Phospho-ATR (Ser428) data with other DDR markers:

  • Upstream sensors (RPA, ATRIP, TOPBP1)

  • Downstream effectors (Phospho-Chk1, Phospho-p53)

  • Parallel pathways (ATM-Chk2 axis)

  • DNA repair markers (γH2AX, 53BP1, Rad51)

• Pathway Visualization Tools:

  • Use pathway mapping software (Ingenuity, Cytoscape) to visualize relationships

  • Create heat maps showing activation patterns across conditions

  • Apply principal component analysis to identify key determinants of response

• Temporal Integration:

  • Develop timeline models of DDR activation

  • Identify sequential activation patterns

  • Determine rate-limiting steps in response pathways

• Functional Correlation:

  • Correlate phosphorylation patterns with functional outcomes (cell cycle arrest, apoptosis, DNA repair efficiency)

  • Establish predictive biomarker signatures for specific outcomes

  • Identify threshold levels associated with cellular decision points

• Single-Cell Approaches:

  • Recognize that population averages may mask important cellular heterogeneity

  • Implement single-cell analyses where possible to capture the full spectrum of responses

  • Identify distinct cellular subpopulations with unique DDR pathway configurations

By integrating these approaches, researchers can move beyond single-marker analysis to develop systems-level understanding of DNA damage response mechanisms.