Phospho-XIAP (Ser87) Antibody

What is XIAP and why is the phosphorylated form at Serine 87 significant?

XIAP (X-linked inhibitor of apoptosis) is a multi-functional protein belonging to the IAP family with E3 ubiquitin-protein ligase activity. It functions as an apoptotic suppressor by inhibiting caspase-3, -7, and -9, and mediates activation of MAP3K7/TAK1, leading to NF-kappa-B activation .

The phosphorylation of XIAP at Serine 87 by Protein Kinase C (PKC) is particularly significant because this post-translational modification makes XIAP resistant to degradation, thereby enhancing its ability to protect cells from apoptosis-inducing stress . This phosphorylation represents a key regulatory mechanism for controlling XIAP's anti-apoptotic function in cellular stress conditions.

What are the primary research applications for Phospho-XIAP (Ser87) antibody?

Phospho-XIAP (Ser87) antibody has been validated for multiple research applications:

The antibody has been particularly useful in tracking the activation state of XIAP in various experimental models involving apoptosis regulation, cancer research, and cellular stress response studies .

How should researchers validate the specificity of Phospho-XIAP (Ser87) antibody?

Phospho-specific antibody validation requires careful controls to ensure specificity for the phosphorylated form:

• Phosphatase treatment control: Treat one sample with lambda phosphatase before immunoblotting to confirm signal loss with the phospho-specific antibody.

• Competing peptide assay: As demonstrated in validation data, competing with phospho-peptide blocks antibody reactivity while non-phosphorylated peptide does not affect signal .

• Positive controls: Use lysates from PDGF-treated NIH/3T3 cells or Anisomycin-treated HepG2 cells, which show increased phosphorylation at Ser87 .

• Known stimulation condition: Compare samples from unstimulated cells versus cells treated with PKC activators, which should increase phosphorylation at Ser87 .

• Cross-validation: Compare results with alternative phospho-XIAP antibodies or phospho-enrichment followed by mass spectrometry to confirm specificity.

What is the optimal protocol for Western blot detection of phosphorylated XIAP?

For optimal detection of phosphorylated XIAP at Ser87 by Western blot:

• Sample preparation: Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate, and β-glycerophosphate) in lysis buffer to preserve phosphorylation status.

• Gel conditions: Use 10% SDS-PAGE for optimal resolution around the 57 kDa observed molecular weight .

• Transfer conditions: Transfer proteins to PVDF membrane at 100V for 90 minutes in cold transfer buffer containing 20% methanol.

• Blocking: Block with 5% BSA (not milk, which contains phosphatases) in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute Phospho-XIAP (Ser87) antibody 1:1000-1:3000 in 5% BSA/TBST and incubate overnight at 4°C .

• Washing: Wash membrane 4 times for 5 minutes each with TBST.

• Secondary antibody: Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour at room temperature.

• Detection: Visualize using enhanced chemiluminescence. Expected molecular weight is approximately 57 kDa (observed) though calculated weight is 60 kDa .

How can Phospho-XIAP (Ser87) antibody be used to investigate the relationship between XIAP phosphorylation and apoptosis resistance?

Investigating XIAP phosphorylation in relation to apoptosis resistance requires multi-faceted experimental approaches:

• Temporal dynamics analysis: Treat cells with apoptosis inducers (e.g., TRAIL, TNF-α, or chemotherapeutics) and monitor Phospho-XIAP (Ser87) levels over time by Western blot to establish correlation with apoptosis resistance.

• PKC modulation: Use PKC activators (PMA) or inhibitors (staurosporine, Gö6983) to manipulate XIAP phosphorylation levels and assess subsequent changes in apoptotic threshold .

• Site-directed mutagenesis: Compare wild-type XIAP with S87A (phospho-null) and S87E (phospho-mimetic) mutants in apoptosis assays to directly assess the functional significance of this phosphorylation site.

• Caspase activity correlation: Perform parallel assessments of caspase-3/7/9 activities alongside Phospho-XIAP (Ser87) levels to establish mechanistic relationships.

• Subcellular fractionation: Use the antibody in combination with subcellular fractionation to determine if phosphorylation affects XIAP localization during apoptotic signaling.

The phosphorylation status can be correlated with ubiquitination levels and proteasomal degradation rates to further elucidate the mechanism of stabilization .

What are the considerations for using Phospho-XIAP (Ser87) antibody in cancer research models?

Cancer research applications require specific considerations:

• Baseline assessment: Establish baseline Phospho-XIAP (Ser87) levels across various cancer cell lines using consistent lysis and detection protocols.

• Correlation with treatment resistance: Analyze phosphorylation levels in treatment-sensitive versus resistant cell subpopulations to identify potential biomarker applications.

• Combination with other IAP family markers: Incorporate parallel assessment of other IAP family members to comprehensively profile the apoptotic network.

• Tissue microarray analyses: When using IHC applications (dilution 1:100-1:300) on tissue microarrays, employ appropriate positive controls and quantification methods .

• Patient-derived xenograft models: Validate antibody cross-reactivity with both human tumor components and mouse stromal elements in PDX models.

• Drug screening applications: Use the antibody to monitor changes in XIAP phosphorylation in response to novel therapeutic compounds targeting the apoptosis pathway.

How can researchers troubleshoot weak or inconsistent signals when using Phospho-XIAP (Ser87) antibody?

When encountering weak or inconsistent signals:

• Phosphorylation preservation: Ensure samples were collected rapidly and maintained at cold temperatures with phosphatase inhibitors throughout processing.

• Stimulation conditions: Verify PKC activation using positive controls such as PDGF-treated NIH/3T3 cells or Anisomycin-treated HepG2 cells .

• Antibody titration: Test a range of antibody dilutions (1:500-1:6000 for WB) to determine optimal concentration for your specific sample type .

• Signal enhancement strategies:

  • Increase protein loading (50-100 μg total protein)

  • Extend primary antibody incubation to overnight at 4°C

  • Use more sensitive detection systems (enhanced ECL or fluorescent secondary antibodies)

• Membrane considerations: PVDF membranes may provide better retention of phosphoproteins compared to nitrocellulose.

• Storage conditions check: Verify antibody has been stored appropriately at -20°C and has not undergone multiple freeze-thaw cycles .

What are the critical controls needed when studying XIAP phosphorylation in complex experimental models?

Complex experimental designs require rigorous controls:

• Phosphorylation-state controls:

  • Lambda phosphatase-treated lysates as negative control

  • PKC activator-treated samples as positive control

• Antibody specificity controls:

  • Pre-absorption with phospho-peptide versus non-phospho-peptide

  • XIAP knockout or knockdown samples to confirm signal specificity

• Technical controls:

  • Total XIAP antibody in parallel to assess changes in total protein levels

  • Phosphorylation-independent loading control (e.g., β-actin)

  • Phosphorylation-dependent positive control (e.g., phospho-ERK following stimulation)

• Biological context controls:

  • Time-course analysis to capture dynamic changes

  • Dose-response relationships for stimulating agents

  • Parallel assessment of functional endpoints (apoptosis markers, caspase activation)

How does XIAP phosphorylation at Ser87 interact with other post-translational modifications of XIAP?

XIAP undergoes multiple post-translational modifications that collectively regulate its function:

• Interaction with ubiquitination: Evidence suggests phosphorylation at Ser87 protects XIAP from ubiquitination and subsequent degradation . Researchers should investigate:

  • Sequential immunoprecipitation experiments to correlate phosphorylation with ubiquitination status

  • Time-course studies following PKC activation to track phosphorylation and subsequent changes in ubiquitination

• S-Nitrosylation interplay: S-Nitrosylation down-regulates XIAP's E3 ubiquitin-protein ligase activity . Potential research questions include:

  • Does phosphorylation at Ser87 affect susceptibility to S-nitrosylation?

  • Are these modifications mutually exclusive or synergistic?

• Other phosphorylation sites: XIAP contains multiple potential phosphorylation sites. Researchers should consider:

  • Comparing Ser87 phosphorylation with other known phosphorylation events

  • Using phospho-proteomic approaches to map the complete phosphorylation profile

• Functional consequence analysis: Combine site-directed mutagenesis with functional assays to determine how Ser87 phosphorylation affects specific XIAP functions:

  • Caspase inhibition capacity

  • E3 ligase activity

  • Protein-protein interactions with binding partners

What are the emerging techniques for studying XIAP phosphorylation dynamics in living cells?

Emerging approaches for studying real-time phosphorylation dynamics include:

• Phospho-specific biosensors: Design FRET-based biosensors that change conformation upon XIAP phosphorylation, allowing real-time monitoring in living cells.

• Proximity ligation assays: Adapt in situ PLA techniques using the Phospho-XIAP (Ser87) antibody together with antibodies against potential interacting partners to visualize interactions specifically with the phosphorylated form.

• Phospho-specific protein complementation: Split fluorescent protein systems that reconstitute only when Phospho-XIAP (Ser87) is recognized by a phospho-binding domain.

• Quantitative phosphoproteomics: Combine SILAC or TMT labeling with phospho-enrichment and mass spectrometry to quantify changes in Ser87 phosphorylation across different experimental conditions.

• In-cell NMR approaches: As highlighted in the PNAS publication (search result ), in-cell destabilization of protein complexes can be detected with advanced NMR techniques, potentially allowing detection of phosphorylation-induced conformational changes.

What are the optimal storage conditions for maintaining Phospho-XIAP (Ser87) antibody activity?

For optimal antibody performance:

• Storage temperature: Store at -20°C for most formulations. Some versions may require -80°C storage .

• Buffer composition: Most commercial preparations contain:

  • PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

  • Some formulations include 0.5% BSA as a stabilizer

• Aliquoting recommendations: For antibodies in glycerol, aliquoting is generally unnecessary for -20°C storage, but may be advisable to avoid repeated freeze-thaw cycles .

• Freeze-thaw cycles: Minimize freeze-thaw cycles; excessive cycles can lead to antibody denaturation and loss of activity .

• Working dilution handling: Diluted working solutions should be prepared fresh and used within 24 hours when kept at 4°C.

• Shelf-life considerations: Most preparations are stable for up to 1 year from date of receipt when stored properly .

How should researchers prepare samples to best preserve XIAP phosphorylation status?

Preserving phosphorylation status requires careful sample handling:

• Rapid processing: Minimize the time between cell harvesting and protein denaturation to prevent phosphatase activity.

• Phosphatase inhibitor cocktail: Include a comprehensive phosphatase inhibitor cocktail in lysis buffers:

  • Sodium fluoride (50 mM) - serine/threonine phosphatase inhibitor

  • Sodium orthovanadate (1 mM) - tyrosine phosphatase inhibitor

  • β-glycerophosphate (10 mM) - serine/threonine phosphatase inhibitor

  • Sodium pyrophosphate (5 mM) - additional phosphatase inhibitor

• Cold chain maintenance: Keep samples cold throughout processing (on ice or at 4°C).

• Denaturing conditions: Use strong denaturing conditions in lysis buffer (1% SDS, boiling) to rapidly inactivate phosphatases.

• Sample storage: Store processed samples at -80°C; avoid repeated freeze-thaw cycles.

• Positive controls: Include known positive controls (e.g., PDGF-treated NIH/3T3 cells) to verify phosphorylation preservation throughout sample processing .

How do different commercial Phospho-XIAP (Ser87) antibodies compare in research applications?

Based on the available search results, several manufacturers offer Phospho-XIAP (Ser87) antibodies with slightly different specifications:

All tested antibodies show reactivity with human, mouse, and rat samples, and most are rabbit polyclonal antibodies purified by affinity chromatography .

Researchers should select the appropriate antibody based on their specific application needs and validate each lot for their experimental system.

What experimental considerations are important when comparing phosphorylation at different sites within XIAP?

When investigating multiple phosphorylation sites within XIAP:

• Temporal regulation patterns: Different sites may be phosphorylated with distinct kinetics following stimulation. Design time-course experiments to capture these differences.

• Kinase specificity: Different kinases target specific sites (e.g., PKC phosphorylates Ser87). Use specific kinase inhibitors to dissect regulation of each site.

• Functional consequences: Different phosphorylation events may affect specific XIAP functions:

  • Caspase binding and inhibition

  • E3 ligase activity

  • Protein-protein interactions

  • Subcellular localization

• Epitope masking issues: Phosphorylation at one site may affect antibody accessibility to nearby sites. Consider using alternative detection methods like mass spectrometry for comprehensive analysis.

• Site interdependence: Phosphorylation at one site may promote or inhibit modifications at other sites. Use phospho-mimetic and phospho-null mutations in combination to study these relationships.

• Pathway-specific regulation: Different cellular pathways may preferentially target specific sites. Compare diverse stimuli (growth factors, stress inducers, apoptotic triggers) for their effects on each phosphorylation site.