Phospho-SMC1A (S957) Recombinant Monoclonal Antibody

What is the biological significance of SMC1A phosphorylation at serine 957?

SMC1A phosphorylation at serine 957 represents a critical regulatory event in the activation of the SMC1A protein, a key component of the cohesin complex. This phosphorylation plays essential roles in multiple cellular processes including DNA damage response, cell cycle regulation, and maintenance of genomic stability. Specifically, SMC1A functions within the cohesin complex to ensure proper sister chromatid cohesion during cell division and participates in DNA repair mechanisms . Phosphorylation at S957 occurs as part of the intra-S-phase DNA damage checkpoint and can be mediated through ATM/NBS1-dependent pathways in response to ionizing radiation, serving as a downstream effector in both ATM/NBS1 and ATR/MSH2 branches of the S-phase checkpoint .

How does Phospho-SMC1A (S957) antibody specificity impact experimental outcomes?

The specificity of Phospho-SMC1A (S957) antibodies directly impacts the reliability and reproducibility of experimental data. These antibodies are designed to recognize only the phosphorylated form of SMC1A at the S957 position, allowing researchers to distinguish between phosphorylated and non-phosphorylated states of the protein . High-specificity antibodies minimize cross-reactivity with other phosphorylated proteins or non-phosphorylated SMC1A, reducing background noise and false positives in Western blots, immunohistochemistry, and other applications. When selecting an antibody, researchers should verify that it has been validated for their specific application and species of interest, as antibody performance can vary between experimental conditions. For optimal results, validation should include positive controls (known phosphorylation-inducing treatments) and negative controls (phosphatase treatments or non-phosphorylatable mutants) .

What is the relationship between SMC1A phosphorylation and the DNA damage response?

SMC1A phosphorylation at S957 functions as a critical mediator in the cellular response to DNA damage. This phosphorylation event is triggered by DNA damage and serves as part of the intra-S-phase checkpoint mechanism. Research has demonstrated that SMC1A becomes phosphorylated at S957 and S966 in an ATM/NBS1-dependent manner following exposure to ionizing radiation . Importantly, this phosphorylation pathway differs depending on the type of DNA damage; while ionizing radiation triggers an ATM-dependent response, other forms of DNA damage such as UV light and hydroxyurea treatment induce SMC1A phosphorylation through ATM-independent mechanisms . SMC1A phosphorylation also enables its interaction with BRCA1, further connecting it to DNA repair pathways . Functionally, these phosphorylation events help prevent damaged DNA from being replicated, allowing time for repair processes to occur and maintaining genomic integrity.

What are the optimal protocols for detecting phosphorylated SMC1A using Western blotting?

For optimal detection of phosphorylated SMC1A using Western blotting, researchers should follow these methodological guidelines:

• Sample preparation: Harvest cells during the appropriate cell cycle phase or following DNA damage induction. Lyse cells in a buffer containing phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) to preserve phosphorylation states.

• Protein separation: Resolve 20-50μg of total protein on a 6-8% SDS-PAGE gel (SMC1A is a large protein of approximately 143 kDa).

• Transfer conditions: Use wet transfer to PVDF membranes at 30V overnight at 4°C for efficient transfer of large proteins.

• Blocking and antibody incubation: Block with 5% BSA (not milk, which contains phosphatases) in TBST. Dilute primary Phospho-SMC1A (S957) antibody according to manufacturer recommendations (typically 1:500 - 1:2000) .

• Detection controls: Include a total SMC1A antibody on parallel blots to normalize phosphorylation signals to total protein levels. Include both positive controls (DNA damage-induced cells) and negative controls (phosphatase-treated lysates).

• Visualization: Use high-sensitivity ECL or fluorescent detection systems for clear visualization of phospho-specific signals .

The molecular weight of phosphorylated SMC1A appears at approximately 143 kDa, though some variation may occur depending on the gel system used and post-translational modifications present.

How can researchers effectively use Phospho-SMC1A (S957) antibodies in immunofluorescence studies?

Effective immunofluorescence detection of phosphorylated SMC1A requires careful attention to sample preparation and staining protocols:

• Fixation method selection: Use 4% paraformaldehyde for 15 minutes at room temperature to preserve phospho-epitopes. Avoid methanol fixation which can cause phospho-epitope loss.

• Permeabilization optimization: Permeabilize with 0.2% Triton X-100 for 10 minutes to allow antibody access while preserving nuclear architecture.

• Blocking strategy: Block with 3-5% BSA containing phosphatase inhibitors to prevent dephosphorylation during processing.

• Antibody validation: Confirm specificity using phosphatase treatments or non-phosphorylatable mutants as negative controls.

• Co-staining approaches: Combine with cell cycle markers (e.g., PCNA for S-phase, histone H3-pS10 for mitosis) to correlate phosphorylation with cell cycle phases. Studies have shown that SMC1A phosphorylation increases during mitosis compared to interphase cells .

• Confocal imaging parameters: Use high-resolution confocal microscopy with Z-stacking to accurately visualize nuclear localization patterns.

For optimal results, researchers should examine multiple microscopic fields and quantify signal intensities across different experimental conditions. Appropriate positive controls include cells treated with DNA-damaging agents like ionizing radiation or hydroxyurea, which induce SMC1A phosphorylation .

What techniques can be employed to validate antibody specificity for phosphorylated SMC1A?

Validating the specificity of Phospho-SMC1A (S957) antibodies is crucial for experimental reliability. Researchers should employ multiple complementary approaches:

• Phosphatase treatment: Treating cell lysates with lambda phosphatase should eliminate antibody reactivity if it's truly phospho-specific.

• Phospho-null mutants: Generate S957A point mutations in SMC1A and confirm the antibody doesn't recognize this non-phosphorylatable form.

• Phosphomimetic controls: Compare detection of wild-type, S957A, and S957D/E (phosphomimetic) SMC1A variants to confirm phospho-specificity.

• Peptide competition assays: Pre-incubate the antibody with phosphorylated and non-phosphorylated peptides surrounding the S957 site. Only the phospho-peptide should block specific binding.

• Kinase and phosphatase modulators: Treat cells with ATM/ATR inhibitors to decrease phosphorylation or with phosphatase inhibitors to increase it, then verify corresponding changes in antibody signal.

• Knockout/knockdown validation: Use SMC1A knockout or knockdown cells as negative controls, confirming loss of the phospho-specific signal .

• Multiple detection methods: Cross-validate findings using different techniques (Western blot, immunofluorescence, ELISA) to ensure consistent results across platforms .

How does the cross-talk between SMC1A acetylation and phosphorylation regulate cellular functions?

The interplay between SMC1A acetylation and phosphorylation represents a sophisticated regulatory mechanism controlling chromosome dynamics and cell cycle progression. Research has revealed a negative correlation between acetylation at K579 and phosphorylation of SMC1A at S957. Specifically:

• Molecular mechanism: SIRT2-mediated deacetylation of SMC1A at K579 promotes its phosphorylation, creating a deacetylation-phosphorylation regulatory axis .

• Cell cycle dynamics: SMC1A K579 acetylation levels decrease during mitosis while phosphorylation levels increase, suggesting temporal regulation through these modifications. Immunofluorescence studies using anti-α-tubulin antibody to visualize the spindle and DAPI for chromosome staining have confirmed this inverse relationship .

• Oxidative stress response: Under oxidative stress conditions (H₂O₂ treatment), acetylmimetic mutation of SMC1A K579 (K579Q) significantly impairs SMC1A phosphorylation compared to wild-type and K579R (deacetylation-mimetic) proteins .

• Functional consequences: Phosphorylation promotes cell proliferation and migration, while K579 acetylation exhibits tumor suppressor properties. The acetylmimetic K579Q mutant significantly inhibits cell growth both in vitro and in xenograft models, displaying lower Ki-67 proliferation indices compared to wild-type or phosphorylation-mimetic variants .

This evidence points to a mechanistic model where SIRT2 functions as an oncogene by deacetylating SMC1A at K579, thereby enhancing its phosphorylation and promoting tumor cell survival under oncogenic stress conditions.

How does Phospho-SMC1A (S957) contribute to cancer development and therapeutic responses?

Phosphorylated SMC1A plays complex roles in cancer biology with significant implications for therapeutic approaches:

• Cancer development: SIRT2-mediated deacetylation promotes SMC1A phosphorylation, allowing cancer cells to overcome mitotic catastrophe and continue proliferating despite oncogenic stress. This mechanism appears particularly important in early-stage carcinomas across various organs .

• Cellular proliferation: Phosphorylated SMC1A promotes cancer cell proliferation and migration, contributing to tumor growth. In xenograft experiments, tumors expressing phosphorylation-mimetic SMC1A mutations (K579Q-DD) developed larger tumors with higher Ki-67 proliferation indices compared to controls .

• Chemotherapeutic responses: The acetylation-phosphorylation status of SMC1A influences sensitivity to common chemotherapeutic agents. Enhanced K579 acetylation (which reduces phosphorylation) significantly increases sensitivity to lower doses of oxaliplatin and 5-fluorouracil in colon cancer models, promoting apoptosis through increased C-PARP and C-Cas3 expression .

• Therapeutic targeting: Inhibition of SIRT2 activity or increasing SMC1A-K579 acetylation induces abnormal chromosome segregation, triggering mitotic catastrophe in cancer cells and enhancing their vulnerability to chemotherapeutic agents. This suggests the SIRT2-SMC1A axis may be a promising therapeutic target .

Research indicates that monitoring Phospho-SMC1A levels may serve as a biomarker for predicting treatment responses, particularly for therapies targeting DNA damage repair pathways or mitotic progression.

What role does Phospho-SMC1A (S957) play in different DNA damage response pathways?

Phospho-SMC1A (S957) functions as a critical effector in multiple DNA damage response pathways, with pathway-specific activation mechanisms:

• Ionizing radiation response: Following exposure to ionizing radiation, SMC1A becomes phosphorylated at S957 through an ATM/NBS1-dependent pathway. This phosphorylation event is part of the intra-S-phase checkpoint mechanism that prevents damaged DNA from proceeding through replication .

• Alternative activation mechanisms: While ionizing radiation triggers ATM-dependent phosphorylation, other forms of DNA damage, including UV light and hydroxyurea treatment, induce SMC1A phosphorylation through ATM-independent mechanisms, possibly via the ATR kinase pathway .

• Dual checkpoint roles: Phosphorylated SMC1A functions as a downstream effector in both the ATM/NBS1 and ATR/MSH2 branches of the S-phase checkpoint system, enabling comprehensive cellular responses to different types of DNA damage .

• BRCA1 interaction network: Phosphorylated SMC1A interacts with BRCA1, integrating it into a broader DNA repair network crucial for maintaining genomic stability. This interaction facilitates recruitment of repair factors to damage sites .

• Sister chromatid cohesion: Beyond checkpoint signaling, phosphorylated SMC1A maintains proper sister chromatid cohesion after DNA damage, ensuring accurate repair through homologous recombination mechanisms .

Understanding these pathway-specific roles of phosphorylated SMC1A provides insights into cellular responses to various genotoxic stressors and may inform strategies for targeting DNA repair deficiencies in cancer therapy.

What are common challenges in detecting Phospho-SMC1A (S957) and how can they be overcome?

Researchers frequently encounter specific challenges when working with Phospho-SMC1A (S957) antibodies. These issues and their solutions include:

• Low signal intensity:

  • Cause: Phosphorylation may be transient or present at low levels under basal conditions.

  • Solution: Induce DNA damage (e.g., ionizing radiation, hydroxyurea) to enhance phosphorylation. Optimize antibody concentration (1:500-1:2000 for Western blot) . Use signal enhancement systems like TSA (tyramide signal amplification).

• High background:

  • Cause: Non-specific binding or phosphatase activity during sample preparation.

  • Solution: Use BSA instead of milk for blocking. Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride) in all buffers. Increase washing duration and frequency. Optimize antibody dilution (1:50-1:200 for IHC-P) .

• Inconsistent results:

  • Cause: Cell cycle variation in phosphorylation levels or phosphatase activity during processing.

  • Solution: Synchronize cells or sort by cell cycle phase. Standardize lysate preparation protocols with immediate denaturation to prevent phosphatase activity.

• Multiple bands in Western blots:

  • Cause: Degradation products, isoforms, or cross-reactivity.

  • Solution: Use freshly prepared samples with protease inhibitors. Validate with phospho-null mutants (S957A) to confirm specificity .

• Weak signal in fixed samples:

  • Cause: Phospho-epitope masking or destruction during fixation.

  • Solution: Optimize fixation time (10-15 minutes in 4% PFA). Consider antigen retrieval methods specific for phospho-epitopes. Avoid methanol fixation.

These methodological adjustments can significantly improve detection sensitivity and specificity when working with Phospho-SMC1A (S957) antibodies.

How can researchers quantitatively analyze Phospho-SMC1A (S957) levels across different experimental conditions?

Accurate quantification of Phospho-SMC1A (S957) requires rigorous methodological approaches:

• Western blot quantification:

  • Normalize phospho-specific signals to total SMC1A protein using parallel blots or sequential probing.

  • Use housekeeping proteins (β-actin, GAPDH) as loading controls.

  • Employ digital imaging systems (e.g., ChemiDoc) with linear dynamic range.

  • Analyze multiple biological replicates (n≥3) using software like ImageJ for densitometry.

• Flow cytometry analysis:

  • Develop dual-staining protocols with total SMC1A and phospho-specific antibodies.

  • Gate populations by cell cycle phases using DNA content dyes.

  • Calculate phosphorylation index as the ratio of phospho-signal to total protein.

• Immunofluorescence quantification:

  • Acquire images under identical exposure settings across samples.

  • Measure nuclear intensity across >100 cells per condition.

  • Correlate with cell cycle markers to account for cell cycle-dependent variation.

  • Employ automated image analysis software for unbiased quantification.

• ELISA-based approaches:

  • Develop sandwich ELISA using capture antibodies against total SMC1A and detection antibodies against phosphorylated epitopes.

  • Generate standard curves using recombinant phosphorylated proteins.

  • Calculate absolute concentrations of phosphorylated protein .

• Mass spectrometry:

  • Use phospho-enrichment strategies (IMAC, TiO₂) before MS analysis.

  • Employ targeted MS approaches (PRM, MRM) for absolute quantification.

  • Integrate peaks corresponding to phosphorylated peptides and normalize to non-phosphorylated counterparts.

These quantitative approaches enable robust comparison of phosphorylation levels across experimental conditions, time points, or treatment groups.

How should researchers interpret changes in SMC1A phosphorylation in the context of cell cycle progression and DNA damage?

Interpreting changes in SMC1A phosphorylation requires careful consideration of cellular context:

• Cell cycle phase considerations:

  • Phosphorylation naturally increases during mitosis compared to interphase .

  • Always normalize comparisons to cells in equivalent cell cycle phases.

  • Consider using cell synchronization or cell cycle markers (PCNA for S-phase, pH3 for mitosis) to contextualize phosphorylation changes.

• Temporal dynamics after DNA damage:

  • SMC1A phosphorylation typically peaks 1-2 hours after ionizing radiation exposure.

  • Kinetics vary by damage type (faster with IR, slower with replication stress).

  • Persistent phosphorylation beyond 24 hours may indicate unresolved damage.

• Pathway-specific interpretation:

  • Determine whether phosphorylation is ATM-dependent (ionizing radiation) or ATM-independent (UV, hydroxyurea) .

  • Compare with other checkpoint markers (γH2AX, Chk1/2 phosphorylation) to assess pathway activation.

• Functional correlation:

  • Increased phosphorylation without cell cycle arrest may indicate checkpoint adaptation.

  • In cancer contexts, elevated phosphorylation may reflect an attempt to overcome genomic instability.

  • The balance between acetylation and phosphorylation affects cellular outcomes - phosphorylation typically promotes proliferation while acetylation can suppress it .

• Experimental validation approaches:

  • Use kinase inhibitors (ATM/ATR inhibitors) to confirm causal relationships.

  • Compare with phospho-null mutants (S957A) to verify functionality.

  • Correlate with downstream phenotypes (cell survival, chromosomal abnormalities) to establish biological significance.

Proper interpretation requires integrating phosphorylation data with other cellular markers and functional outcomes to establish meaningful correlations and causal relationships.

What are the comparative advantages of different Phospho-SMC1A (S957) antibody formats?

Different antibody formats offer distinct advantages depending on research applications:

Selecting the appropriate format should be guided by the specific experimental requirements, with recombinant monoclonal antibodies generally offering the best combination of consistency and specificity for quantitative applications .

How does the specificity and sensitivity of Phospho-SMC1A (S957) antibodies vary across different applications?

Antibody performance varies significantly across different experimental techniques:

For optimal results, researchers should validate each antibody for their specific application, as performance can vary substantially between techniques. The choice of detection system also significantly impacts sensitivity, with enhanced chemiluminescence or fluorescent secondary antibodies improving detection limits in most applications .

What considerations are important when designing experiments using Phospho-SMC1A (S957) antibodies across multiple species?

Cross-species experimentation with Phospho-SMC1A (S957) antibodies requires careful planning:

• Sequence conservation analysis:

  • The S957 phosphorylation site is well-conserved across mammals but may vary in non-mammalian species.

  • Human and mouse sequences show high homology around the S957 site, making many antibodies cross-reactive between these species .

• Validation requirements for each species:

  • Even with conserved epitopes, antibody performance must be validated in each species.

  • Phospho-null mutants (S957A) should be generated in the species of interest when possible.

  • Phosphatase treatments should confirm specificity in each new species model.

• Application-specific considerations:

  • Western blotting may show cross-reactivity across species due to denatured epitopes.

  • Native-state applications (IP, IF) often show more species restriction.

• Species-dependent regulatory differences:

  • While the phosphorylation site may be conserved, upstream kinases and regulatory mechanisms may differ.

  • DNA damage-induced phosphorylation kinetics may vary between species.

• Commercial antibody selection:

  • Choose antibodies validated for multiple species when available.

  • Most commercial Phospho-SMC1A (S957) antibodies are validated for human samples, with some showing reactivity to mouse and rat .

• Controls for cross-species applications:

  • Include species-matched positive controls (treated samples known to express phosphorylated protein).

  • Perform side-by-side comparisons with human samples when using human-optimized antibodies.

These considerations ensure that experimental findings using Phospho-SMC1A (S957) antibodies can be reliably interpreted across different model organisms and translated to human biology.