PTTG1 Antibody

What is PTTG1 and what cellular functions does it regulate?

PTTG1 (also known as securin) functions as a key regulator of sister chromatid separation during cell division. It plays critical roles in normal physiological conditions, particularly in regulating human embryonic stem cells and maintaining the stem cell compartment . As a multifunctional protein, PTTG1 contains 202 amino acids with no significant similarity to other known proteins . Its expression is highly restricted in normal tissues, being predominantly expressed in testis with minimal expression in normal ovary and breast tissues .

What is the molecular weight of native and tagged PTTG1 protein in Western blotting applications?

The molecular weight profile of PTTG1 varies depending on its form:

• Native PTTG1 protein: Approximately 28 kDa in cell extracts

• GST-PTTG1 recombinant protein: 52 kDa

• PTTG1-GFP chimeric construct: 56 kDa

These differences are important when validating antibody specificity in Western blotting applications, as the detection of bands at incorrect molecular weights could indicate non-specific binding or protein degradation.

What alternative nomenclature exists for PTTG1 in scientific literature?

PTTG1 is referenced under several alternative names in research papers and databases:

• HPTTG

• PTTG

• Securin

• EAP1

• TUTR1

• ESP1-associated protein 1

Researchers should be aware of these alternative designations when conducting literature searches to ensure comprehensive coverage of relevant studies.

How does PTTG1 expression change during tumor progression?

PTTG1 shows a consistent pattern of upregulation during tumor progression. Studies examining normal ovary (NO) samples compared to benign tumor (BN), borderline tumor (BL), and high-grade tumors (HG) have documented a graded increase in PTTG1 expression . While there is considerable inter-patient variation, this pattern of progressive upregulation makes PTTG1 a potential biomarker for tumor progression. The expression increase is often concurrent with upregulation of cancer stem cell markers like CD133 and CD24 .

What is the relationship between PTTG1 and cancer stem cells?

PTTG1 has been demonstrated to co-localize with multiple stem cell/cancer stem cell markers including:

• ALDH1

• CD34

• CD44

• LGR5

• NANOG

• OCT4

• SSEA4

This co-localization occurs in both normal ovarian samples and ovarian tumors at various stages. Notably, PTTG1 is highly expressed in ALDH1+ cancer stem cells compared to ALDH1- cells, suggesting it may function as a previously overlooked marker for stem cells and cancer stem cells . Research indicates PTTG1 may directly regulate self-renewal mechanisms and epithelial-mesenchymal transition (EMT) pathways in cancer stem cells.

What experimental models are best for studying PTTG1 function in tumorigenesis?

Both in vitro and in vivo models have proven valuable for PTTG1 research:

What critical parameters should researchers evaluate when selecting a PTTG1 antibody?

When selecting a PTTG1 antibody, researchers should consider:

• Epitope specificity: Some antibodies target the full-length protein (AA 1-202), while others target specific epitopes

• Validated applications: Confirm the antibody has been validated for your specific application (WB, IHC, IP, etc.)

• Host species: Consider compatibility with other antibodies for dual-labeling experiments

• Reactivity: Ensure the antibody recognizes PTTG1 from your species of interest (human, mouse, etc.)

• Published validation: Review literature using the antibody to assess reliability and reproducibility

• Clonality: Monoclonal antibodies offer consistency, while polyclonal antibodies may provide increased sensitivity

What controls are essential for validating PTTG1 antibody specificity?

Rigorous validation controls include:

• Positive tissue controls: Testis tissue (known to express PTTG1)

• Negative tissue controls: Normal breast or ovary tissue (minimal PTTG1 expression)

• Recombinant protein: Purified PTTG1 protein as a positive control for Western blotting

• Genetically modified cells: PTTG1 knockout/knockdown cells as negative controls

• Overexpression systems: Cells transfected with PTTG1 expression constructs as positive controls

• Peptide competition: Pre-incubation of antibody with immunizing peptide should abolish specific signal

How can researchers optimize antibody dilution for different experimental applications?

How can dual immunofluorescence be optimized for co-localization studies of PTTG1 with stem cell markers?

For optimal dual immunofluorescence co-localization:

• Fixation method: Use 4% paraformaldehyde for 15-20 minutes to preserve epitope accessibility

• Permeabilization: 0.1-0.3% Triton X-100 for intracellular antigens

• Blocking: Use 5-10% serum from secondary antibody host species for 1 hour

• Primary antibodies: Apply PTTG1 antibody alongside stem cell marker antibodies (ALDH1, CD44, etc.)

• Secondary antibodies: Use species-specific secondaries with distinct fluorophores

• Nuclear counterstain: DAPI for nuclear localization context

• Controls: Single-antibody controls to verify absence of cross-reactivity

• Imaging: Confocal microscopy for accurate co-localization analysis

Research has successfully demonstrated co-localization of PTTG1 with multiple stem cell markers in both ovarian surface epithelium and cortex regions in normal ovary and ovarian tumors .

What approaches can researchers use to study PTTG1's role in the regulation of cancer stem cell populations?

To investigate PTTG1's regulatory functions in cancer stem cells:

• Gene expression manipulation:

  • siRNA or shRNA knockdown of PTTG1 to assess loss-of-function effects

  • CRISPR-Cas9 knockout for complete gene elimination

  • Overexpression studies using transfection of PTTG1 expression constructs

• Functional assays:

  • Sphere formation assays to assess self-renewal capacity

  • Colony formation assays for clonogenicity

  • Invasion/migration assays to evaluate EMT properties

  • In vivo tumor initiation studies with limiting dilution analysis

• Molecular analysis:

  • qRT-PCR for stem cell markers and self-renewal pathway genes

  • Western blotting for protein expression changes

  • ChIP-seq to identify PTTG1 binding sites on chromatin

  • RNA-seq for global transcriptional changes

Studies have shown that PTTG1 manipulation affects the expression of self-renewal mechanisms and EMT-related genes in cancer stem cells, suggesting its critical role in maintaining stemness properties .

What strategies are effective for studying PTTG1 post-translational modifications and protein interactions?

How can researchers address non-specific binding issues with PTTG1 antibodies in Western blotting?

To minimize non-specific bands in Western blots:

• Optimize blocking conditions: Try different blocking agents (5% milk, 5% BSA, or commercial blockers)

• Increase washing stringency: More frequent washes with higher TBST concentration (0.1-0.3% Tween-20)

• Titrate antibody concentration: Determine minimum effective concentration through serial dilutions

• Use PVDF membranes: May provide better signal-to-noise ratio than nitrocellulose for some antibodies

• Include protein extraction controls: Use protease inhibitors to prevent degradation products

• Try alternative antibodies: Test antibodies targeting different epitopes

• Use gradient gels: Better separation of proteins with similar molecular weights

• Optimize transfer conditions: Adjust time and voltage for complete transfer of protein

What are the most effective antigen retrieval methods for PTTG1 immunohistochemistry?

Research has shown that proper antigen retrieval is critical for detecting PTTG1 in various tumor tissues, with heat-induced methods generally providing superior results .

How can researchers interpret discrepancies between PTTG1 protein levels and mRNA expression?

When facing discrepancies between PTTG1 protein and mRNA levels:

• Consider post-transcriptional regulation:

  • microRNA-mediated suppression of translation

  • RNA-binding protein effects on mRNA stability

  • Alternative splicing generating different isoforms

• Evaluate post-translational regulation:

  • Protein stability differences (proteasomal degradation)

  • Subcellular localization affecting antibody accessibility

  • Post-translational modifications altering epitope recognition

• Technical considerations:

  • Antibody specificity for particular isoforms

  • Sample preparation differences between protein and RNA extraction

  • Tissue heterogeneity and sampling differences

Research has noted high variation in PTTG1 expression levels between patients, suggesting complex regulatory mechanisms beyond transcriptional control .

How might PTTG1 serve as a therapeutic target in cancer treatment?

PTTG1 shows significant potential as a therapeutic target based on several lines of evidence:

• Knockdown effects: Various methods of PTTG1 downregulation have been shown to inhibit ovarian cell proliferation and suppress tumor growth in nude mice

• Stem cell targeting: PTTG1's expression in cancer stem cells suggests targeting it could eliminate the tumor-initiating population

• Signaling pathway modulation: PTTG1 mediates AKT activation, which is implicated in stemness and EMT properties of cancer cells

• Differential expression: The minimal expression in most normal tissues compared to high expression in tumors provides a therapeutic window

Future therapeutic approaches may include siRNA-based therapies, small molecule inhibitors of PTTG1 or its interaction partners, and antibody-drug conjugates targeting PTTG1-expressing cells.

What novel methodologies are emerging for studying PTTG1 in single-cell and spatial contexts?

Emerging technologies with potential for PTTG1 research include:

• Single-cell proteomics: Measuring PTTG1 protein levels in individual cells to assess heterogeneity

• Spatial transcriptomics: Mapping PTTG1 mRNA expression with spatial resolution in tissue sections

• Imaging mass cytometry: Multiplexed protein detection including PTTG1 with spatial information

• CODEX multiplexed imaging: Simultaneous visualization of PTTG1 with dozens of other markers

• Live-cell imaging with fluorescent PTTG1 reporters: Monitoring dynamic changes in expression

• Organoid models: 3D culture systems to study PTTG1 in more physiologically relevant contexts

These approaches could reveal new insights into PTTG1's spatial distribution within the tumor microenvironment and its relationship to specific cellular niches.

How can researchers integrate PTTG1 data with multi-omics approaches in cancer research?

Integrative multi-omics strategies for PTTG1 research:

• Correlate PTTG1 protein expression with:

  • Genomic alterations (mutations, CNVs)

  • DNA methylation patterns at the PTTG1 promoter

  • Chromatin accessibility at PTTG1 regulatory regions

  • Global transcriptomic profiles

  • Metabolomic signatures

• Data integration methods:

  • Network analysis to identify PTTG1-centered regulatory networks

  • Machine learning approaches to predict PTTG1-high tumor phenotypes

  • Systems biology modeling of PTTG1 pathway interactions

  • Patient stratification based on PTTG1 expression and multi-omics profiles

• Clinical correlations:

  • Integrate PTTG1 expression with treatment response data

  • Develop predictive biomarker signatures incorporating PTTG1

  • Correlate with immune infiltration patterns and immunotherapy response

These integrative approaches could provide comprehensive understanding of PTTG1's role in tumor biology and identify novel therapeutic strategies targeting PTTG1-dependent pathways.