TP53I11 Antibody, FITC conjugated
Description
2.1. Immunofluorescence (IF)
TP53I11-FITC antibodies enable subcellular localization studies. For example:
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Co-localization with Actin: TP53I11 interacts with the actin cytoskeleton in endothelial cells, as demonstrated by IF and co-immunoprecipitation .
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Hypoxia Studies: FITC-conjugated antibodies help visualize TP53I11 upregulation under hypoxia via HIF2A signaling in human umbilical vein endothelial cells (HUVECs) .
2.2. Functional Studies in Cancer
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Angiogenesis: TP53I11 promotes endothelial cell sprouting and tubule formation, critical for tumor angiogenesis .
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Clinical Correlations: Elevated TP53I11 correlates with advanced tumor stages in gastric cancer and reduced survival in breast cancer .
3.1. Role in Tumor Angiogenesis
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Mechanism: TP53I11 enhances GTPase activity and actin cytoskeleton remodeling in endothelial cells, facilitating blood vessel formation .
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Therapeutic Implications: High TP53I11 expression in BRCA, KIRP, and UVM tumors predicts poor prognosis, suggesting its utility as a biomarker .
3.2. Dual Role in Cancer
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Target Background Gene References and Functions
- PIG11 is considered a potential liver tumor suppressor gene. PMID: 19096915
- As a downstream target of p53, PIG11 is involved in the apoptosis of gastric cancer cells. PMID: 12883691
- Overexpression of PIG11 can induce cell apoptosis at low levels and enhance the apoptotic effects of arsenic trioxide. PMID: 15225615
- Jasmonates can overcome drug resistance induced by p53 mutations. PMID: 16170329
- The PIG11 protein may play a significant role in the regulation of apoptosis by interacting with other biological molecules, offering a novel perspective to explore the potential function of PIG11 in vivo. PMID: 17482569
Q&A
How should researchers validate the specificity of TP53I11 antibodies (FITC conjugated) in Western blotting and immunofluorescence?
Validation requires a multi-step approach:
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Positive/Negative Controls: Use cell lines with confirmed TP53I11 expression (e.g., p53-activated models) and knockout/knockdown systems. For example, irradiated MOLM13 cells (wild-type TP53) show upregulated TP53I11, while HL60 cells (TP53-/-) serve as negative controls .
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Cross-Reactivity Checks: Test antibody reactivity against homologous proteins (e.g., murine TP53I11 homologs) using species-specific lysates. Source highlights reactivity in human, mouse, and rat tissues, necessitating species-matched controls.
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Blocking Peptide Competition: Pre-incubate the antibody with the immunogen peptide (e.g., residues 71–120 for ABIN1533484 ) to confirm signal loss in Western blots.
Table 1: Recommended Validation Cell Lines
| Cell Line | TP53 Status | TP53I11 Expression | Application |
|---|---|---|---|
| MOLM13 | Wild-type | Inducible (post-irradiation) | Positive control |
| HL60 | Null | Undetectable | Negative control |
| OV90 | Mutant | Constitutively high | Mutant studies |
What experimental conditions optimize FITC-conjugated TP53I11 antibody performance in live-cell imaging?
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Fixation Permeabilization: Use ice-cold methanol (100%, 10 min) for optimal epitope retention, as formaldehyde may mask the AA 71–120 epitope .
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Signal-to-Noise Ratio: Titrate antibody concentrations between 1:50–1:200 (see source ) and use blocking buffers with 5% BSA to reduce nonspecific binding.
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Quenching: Add 0.1% sodium borohydride to neutralize autofluorescence in fixed cells.
How can TP53I11 antibody cross-reactivity with mutant p53 isoforms confound apoptosis assays, and how is this resolved?
Mutant p53 isoforms (e.g., R175H, R248Q) may aberrantly bind TP53I11 promoters, leading to false-positive signals in chromatin immunoprecipitation (ChIP). Mitigation strategies include:
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Isoform-Specific Knockdown: Use siRNA targeting mutant p53 in parallel with wild-type p53 models.
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Epitope Mapping: Employ antibodies targeting non-overlapping regions (e.g., N-terminal vs. internal epitopes). Source demonstrates that DO-7 (epitope: aa 37–45) avoids cross-reactivity with C-terminal mutants.
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Competitive ELISA: Preclear lysates with unconjugated TP53I11 antibodies to isolate mutant-specific interactions .
What technical considerations apply when integrating FITC-TP53I11 antibodies into mass cytometry panels?
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Metal Isotope Selection: Conjugation with high-sensitivity isotopes (e.g., 153Eu) improves detection of low-abundance TP53I11 compared to 175Lu .
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Signal Compensation: Use a “minus FITC” control tube to correct for spectral overlap in CyTOF panels.
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Multiplexing Limits: Limit panels to ≤3 FITC-conjugated antibodies to avoid channel saturation.
Table 2: Mass Cytometry Optimization Parameters
| Parameter | Recommendation | Rationale |
|---|---|---|
| Antibody Concentration | 0.5–1.0 µg/mL | Balances specificity and signal intensity |
| Incubation Time | 30 min at 4°C | Minimizes internalization |
| Isotope | 153Eu | 40% higher signal than 175Lu in Helios systems |
How do researchers reconcile contradictory data on TP53I11’s role in apoptosis across studies?
Discrepancies often arise from:
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Cell Type Variability: TP53I11 overexpression induces apoptosis in epithelial cells but not fibroblasts . Use tissue-specific models (e.g., OV90 for ovarian cancer).
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Antibody Batch Differences: Polyclonal preparations (e.g., source vs. ) may recognize distinct epitopes. Validate using independent methods (e.g., CRISPR-Cas9 knockout).
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Post-Translational Modifications: Acetylation at K382 (detected by REA529 in source ) alters TP53I11-p53 binding. Include acetylation/mutation controls.
What workflow resolves low signal intensity in TP53I11 immunofluorescence despite high antibody concentrations?
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Epitope Accessibility: Switch to antigen retrieval with citrate buffer (pH 6.0, 95°C, 20 min).
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Conjugate Stability: Check FITC degradation using absorbance at 494 nm; replace if OD < 0.5.
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Alternative Clonality: Compare polyclonal (source ) vs. monoclonal antibodies; the latter reduces background in dense tissues.
How are TP53I11-p53 binding affinities quantified in co-immunoprecipitation assays?
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Radioimmunoassay (RIA): Immobilize T-antigen-p53 complexes on PAb419-coated plates, then compete with FITC-TP53I11 (source ).
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Dissociation Constant (Kd): Calculate via Scatchard plot using iodinated p53 (e.g., 125I-PAb421) at 0.1–10 nM concentrations.
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Confounders: Truncated TP53I11 isoforms (e.g., Δ71–120) lack binding domains, requiring SDS-PAGE to confirm full-length protein .
