PTTG1IP Antibody

What is PTTG1IP and why is it important in cancer research?

PTTG1IP/PBF is a proto-oncogene that was first identified through its ability to bind to the human securin PTTG1 . It plays critical roles in:

• Facilitating the shift of securin from cell cytoplasm to nucleus, allowing interaction between separase and securin

• Regulating p53 activity, decreasing its stability by enhancing ubiquitination

• Metaphase-anaphase transition of the cell cycle through activation of securin

• Thyroid biology, including regulation of the sodium iodide symporter

Its importance in cancer research stems from its overexpression in various tumors (thyroid, breast, and colorectal cancers) and association with poorer oncological outcomes, including decreased disease-specific survival and increased tumor recurrence .

What molecular characteristics of PTTG1IP should researchers be aware of when selecting antibodies?

When selecting PTTG1IP antibodies, researchers should consider:

• Protein size: Human PTTG1IP has a calculated molecular weight of 20.3 kDa (180 amino acids), but is observed at approximately 26 kDa in experimental settings due to post-translational modifications

• Post-translational modifications: PTTG1IP undergoes glycosylation, which affects its molecular weight and possibly its function

• Subcellular localization: PTTG1IP is found in the membrane, nucleus, and cytoplasm

• Epitope regions: Antibodies targeting different regions (N-terminal, C-terminal, internal) are available and may yield different results depending on the research question

• Species cross-reactivity: Many antibodies react with human, mouse, and rat PTTG1IP, but this should be verified for your specific model system

What are the optimal applications for PTTG1IP antibody detection in cancer research?

Based on validated research applications, PTTG1IP antibodies have been successfully used in:

The choice of application should align with your specific research question. For prognostic studies in clinical samples, IHC is well-validated . For mechanistic studies examining protein interactions, IP followed by Western blot is appropriate .

What protocols should be followed for optimal immunohistochemical detection of PTTG1IP in tissue samples?

For optimal IHC detection of PTTG1IP in tissue samples:

• Deparaffinization of formalin-fixed paraffin-embedded sections

• Epitope retrieval: Use of CC2 buffer for 20 minutes or TE buffer at pH 9.0 (alternatively, citrate buffer at pH 6.0)

• Primary antibody incubation: 20 minutes at 37°C using a 1:20-1:200 dilution (antibody-dependent)

• Detection system: HRP-based detection systems such as OmniMap HRP and ChromoMap DAB kits have been validated

• Scoring system: Immunopositivity can be scored based on percentage of positive cells (0: negative, 1+: <10%, 2+: 10-50%, 3+: ≥50%)

For prognostic studies, attention to both the intensity and subcellular localization of staining is essential, as PTTG1IP negativity has been associated with poorer outcomes in breast cancer .

How can researchers effectively study the interaction between PTTG1IP and p53 in cancer models?

To study PTTG1IP-p53 interactions:

• Co-immunoprecipitation (Co-IP):

  • Use anti-PTTG1IP antibody (0.5-4.0 μg) for immunoprecipitation from 1.0-3.0 mg of total protein lysate

  • Perform Western blot with anti-p53 antibody to detect complex formation

• Proximity Ligation Assay (PLA):

  • This technique has been validated for detecting PTTG1IP-p53 interactions in thyroid cells

  • Use specific primary antibodies against PTTG1IP and p53, followed by oligonucleotide-linked secondary antibodies

• Stability assays:

  • Treat cells with anisomycin to block protein synthesis

  • Monitor p53 degradation over time (e.g., 0-120 minutes) in the presence of different levels of PTTG1IP

  • Research has shown that PBF overexpression accelerates p53 degradation while PBF depletion increases p53 stability

• Ubiquitination assays:

  • To determine if PTTG1IP affects p53 ubiquitination, which appears to be dependent on the E3 ligase activity of Mdm2

What approaches can be used to investigate PTTG1IP mutations and their functional consequences?

To investigate PTTG1IP mutations:

• Protein stability assessment:

  • Perform anisomycin half-life studies to compare mutant protein stability to wild-type PTTG1IP

  • C51R and R140W mutations have shown altered protein stability

• Subcellular localization analysis:

  • Use immunofluorescence with epitope-tagged mutant constructs

  • Different mutations show distinct localization patterns (e.g., C51R confined to the endoplasmic reticulum, R140W apparent in the Golgi apparatus)

• Functional assays:

  • Migration assays: C51R and R140W mutations lose capacity to induce cellular migration

  • Invasion assays: These mutations significantly reduce cell invasion

  • Colony formation and soft agar assays: Unlike wild-type PTTG1IP, C51R and R140W mutants were unable to elicit significant colony formation or anchorage-independent growth

• Glycoprotein processing analysis:

  • Mutations like C51R and G106R appear to prevent the full processing of this glycoprotein

How should PTTG1IP immunoexpression be evaluated for prognostic studies in breast cancer?

For evaluating PTTG1IP as a prognostic marker in breast cancer:

• Scoring methodology:

  • Use a 4-tier scoring system based on percentage of positive cells:

    • Score 0: Total PTTG1IP-negativity

    • Score 1+: Weak staining (<10% of cancer cells)

    • Score 2+: Moderate staining (10-50% of cancer cells)

    • Score 3+: Diffuse staining (≥50% of cancer cells)

• Combined marker analysis:

  • Evaluate PTTG1IP together with securin immunoexpression for greater prognostic power

  • PTTG1IP-negativity combined with high securin immunoexpression indicates a high risk of breast cancer death (HR = 2.5, p < 0.0001)

  • This combination resulted in up to 14-year survival difference in breast cancer patients

• Correlation with established prognosticators:

  • PTTG1IP-immunopositivity is inversely related to aggressive disease features

  • Analyze in context of tumor size, axillary lymph node metastasis, histological grade, and intrinsic classification

• Triple-negative breast carcinomas:

  • Evaluate subcellular location of securin, which has potential prognostic value in this subgroup (p = 0.052)

What is the relationship between PTTG1IP expression and immune infiltration in cancer?

Research has identified correlations between PTTG1IP expression and immune infiltration:

• Methodology for assessing immune correlation:

  • Use tumor immune cell estimation resource (TIMER) database or similar tools to assess correlation between PTTG1IP expression and tumor-infiltrating immune cells

  • Employ Spearman's rank-correlation coefficient for correlation analysis

  • Use Wilcoxon rank sum test to compare infiltration between low and high PTTG1IP expression groups

• Specific immune cell correlations:

  • PTTG1IP expression has been correlated with six types of immune cells: B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells

  • This correlation suggests PTTG1IP may be a promising target for immunotherapy approaches

• Prognostic implications:

  • Combined analysis of PTTG1IP expression and immune infiltration patterns provides enhanced prognostic value

  • This relationship has been particularly studied in epithelial ovarian cancer (EOC)

What are common challenges in PTTG1IP antibody detection and how can they be addressed?

Common challenges and solutions include:

• Antibody specificity issues:

  • Problem: Non-specific binding leading to false positive results

  • Solution: Validate antibody specificity using PTTG1IP knockdown/knockout controls and appropriate blocking procedures

• Variable molecular weight detection:

  • Problem: PTTG1IP may appear at different molecular weights (calculated: 20 kDa; observed: 26 kDa)

  • Solution: Account for post-translational modifications, particularly glycosylation; use appropriate positive controls

• Subcellular localization variability:

  • Problem: PTTG1IP localizes to multiple cellular compartments (membrane, nucleus, cytoplasm)

  • Solution: Use cell fractionation methods to confirm localization patterns and consider co-staining with organelle-specific markers

• Epitope masking in fixed tissues:

  • Problem: Formalin fixation may mask epitopes

  • Solution: Optimize antigen retrieval methods (TE buffer pH 9.0 or alternatively citrate buffer pH 6.0)

How can researchers distinguish between PTTG1IP and related proteins in experimental settings?

To ensure specific detection of PTTG1IP:

• Antibody selection:

  • Choose antibodies that detect endogenous levels of total PTTG1IP protein

  • Verify antibody specificity using appropriate controls

• Western blot optimization:

  • Use proper molecular weight markers to distinguish PTTG1IP (observed at ~26 kDa) from related proteins

  • Include positive controls such as A375, HEK-293, or HeLa cell lysates that have been validated for PTTG1IP expression

• Double-staining approaches:

  • For co-localization studies with securin, use immunofluorescence double-staining methods

  • This approach has revealed that cancer cells exhibiting PTTG1IP-positivity show nuclear securin expression, while in absence of PTTG1IP, securin expression shows a shift towards cytoplasmic location

• Genetic approaches:

  • Use siRNA or shRNA targeting PTTG1IP to validate antibody specificity

  • Depleting PBF in human primary thyrocytes has been shown to increase radioiodine uptake, providing a functional readout