PTTG1 Antibody, Biotin conjugated

What is PTTG1 and what are its key biological functions?

PTTG1, also known as Securin, is a multifunctional protein that plays critical roles in cellular processes. It functions primarily as a global transcription factor that specifically modulates the G1/S-phase transition of the cell cycle . PTTG1 has been identified as an Esp1-associated protein (EAP1) and is also referred to as tumor-transforming protein 1 (hPTTG) .

What are the key specifications of commercially available PTTG1 Antibody, Biotin conjugated?

The PTTG1 Antibody, Biotin conjugated is a polyclonal antibody raised in rabbits against recombinant Human Securin protein (amino acids 2-202) . This antibody has the following specifications:

• Host Species: Rabbit

• Clonality: Polyclonal

• Isotype: IgG

• Immunogen: Recombinant Human Securin protein (2-202AA)

• Conjugate: Biotin

• Species Reactivity: Human

• Applications: Primarily ELISA, though related PTTG1 antibodies have applications in Western Blot (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF)

• Purification Method: >95% Protein G purified

• Storage Buffer: Preservative: 0.03% Proclin 300; Constituents: 50% Glycerol, 0.01M PBS, pH 7.4

• Form: Liquid

• UniProt ID: O95997

What is the optimal storage protocol for maintaining antibody activity?

For optimal preservation of antibody activity, PTTG1 Antibody, Biotin conjugated should be stored at -20°C or -80°C upon receipt . It is crucial to avoid repeated freeze-thaw cycles as these can significantly compromise antibody performance .

For working solutions, aliquoting into smaller volumes prior to freezing is recommended to minimize the need for repeated thawing of the stock solution. When handling the antibody, researchers should work quickly and keep the antibody on ice when in use. The antibody is supplied in a buffer containing 50% glycerol, which helps maintain stability during freezing and prevents complete solidification at -20°C .

What experimental applications is this antibody optimized for?

The PTTG1 Antibody, Biotin conjugated is primarily optimized for ELISA applications . The biotin conjugation allows for enhanced sensitivity in detection systems utilizing streptavidin-based amplification. While the product specifications specifically mention ELISA, related PTTG1 antibodies in the same product line have demonstrated utility in additional applications including:

• Western Blotting (WB)

• Immunohistochemistry (IHC)

• Immunoprecipitation (IP)

• Immunocytochemistry (ICC)

• Immunofluorescence (IF)

• Flow Cytometry (FACS)

When adapting this biotin-conjugated antibody for applications beyond ELISA, researchers should perform careful validation studies, including appropriate positive and negative controls to ensure specificity and sensitivity in their particular experimental system.

How does PTTG1 interact with Sp1 to regulate gene expression in cell cycle control?

PTTG1 functions as a transcriptional regulator through its interaction with the transcription factor Sp1, specifically in modulating the G1/S phase transition . Research using co-immunoprecipitation has demonstrated that PTTG1 physically associates with Sp1 in multiple cell lines, including JEG-3 and HCT116 .

The interaction domains have been mapped using His-tag pulldown assays, revealing that the N-terminus of PTTG1 (amino acids 1-120) binds to the C-terminus of Sp1 (amino acids 401-785) . This physical interaction appears to enhance Sp1's DNA binding activity, as demonstrated through electrophoretic mobility shift assays (EMSA) .

Sequential Chromatin Immunoprecipitation (ChIP) experiments have shown that PTTG1 and Sp1 co-localize on the promoter of cyclin D3, a critical regulator of the G1/S phase transition . This co-occupancy suggests a cooperative function in transcriptional regulation of cell cycle genes. Mechanistically, while the PTTG1 N-terminus is responsible for binding to Sp1, the C-terminus of PTTG1 appears to be important for modulating Sp1's activity, enhancing its binding to target DNA motifs .

For researchers investigating these interactions, it is recommended to employ multiple complementary approaches, including co-immunoprecipitation, ChIP, sequential ChIP, and reporter gene assays to thoroughly characterize the functional consequences of this interaction in specific cellular contexts.

What are optimal protocols for using PTTG1 Antibody in ChIP and sequential ChIP experiments?

When utilizing PTTG1 Antibody in Chromatin Immunoprecipitation (ChIP) experiments, researchers should consider the following protocol optimizations:

ChIP Protocol Considerations:

Sequential ChIP (Re-ChIP) Protocol:
For investigating co-occupancy of PTTG1 and Sp1 on target promoters, sequential ChIP can be performed as demonstrated in previous research :

• Perform first-round ChIP with Sp1 antibody following standard protocols.

• After elution of the Sp1-bound chromatin (but before reversal of crosslinks), use this material as the input for a second round of ChIP using the PTTG1 antibody.

• PCR analysis of the final immunoprecipitated DNA can reveal co-occupancy of both factors on specific genomic regions, such as the cyclin D3 promoter .

For the biotin-conjugated PTTG1 antibody, researchers can leverage streptavidin-based capture systems for the immunoprecipitation step, potentially increasing sensitivity and reducing background.

How can PTTG1 Antibody be optimized for detecting endogenous versus overexpressed PTTG1 in cancer research?

Detection of endogenous versus overexpressed PTTG1 presents different challenges that require specific optimization strategies:

For Endogenous PTTG1 Detection:

• Sensitivity Considerations: Endogenous PTTG1 may be expressed at low levels in normal tissues. The biotin-conjugated antibody offers enhanced sensitivity through signal amplification with streptavidin-HRP or streptavidin-fluorophore conjugates.

• Specificity Validation: Confirm antibody specificity using PTTG1 knockout or knockdown controls. This is particularly important when studying tissues with naturally low PTTG1 expression.

• Subcellular Localization: PTTG1 can localize to both nuclear and cytoplasmic compartments. For immunofluorescence applications, careful subcellular fractionation and separate analysis of nuclear versus cytoplasmic fractions may be necessary.

For Overexpressed PTTG1 in Cancer Samples:

• Dynamic Range: In cancer tissues where PTTG1 is highly overexpressed, ensure your detection system has sufficient dynamic range to avoid signal saturation. Dilution series experiments are recommended.

• Quantification Methods: For comparing PTTG1 levels across cancer samples, consider using quantitative approaches such as quantitative Western blotting or flow cytometry with appropriate calibration standards.

• Correlation with Clinical Parameters: When studying cancer samples, correlate PTTG1 expression levels with clinical parameters, as PTTG1 overexpression has been associated with tumor development, size, and malignancy in various cancers .

For both scenarios, titration of the primary antibody concentration is essential to determine the optimal working concentration for each specific application and sample type.

What experimental approaches can elucidate the relationship between PTTG1 expression and cell cycle regulation?

To investigate PTTG1's role in cell cycle regulation, researchers can employ several complementary experimental approaches:

1. Cell Synchronization and Cell Cycle Analysis:

• Synchronize cells at different cell cycle phases (G0/G1, S, G2/M) using methods such as serum starvation, double thymidine block, or nocodazole treatment.

• Analyze PTTG1 expression and localization across the cell cycle using the biotin-conjugated PTTG1 antibody in combination with flow cytometry and immunofluorescence.

• Correlate PTTG1 levels with cell cycle markers (e.g., cyclin D3, cyclin B1) to establish temporal relationships.

2. ChIP-Seq Analysis:

• Perform genome-wide mapping of PTTG1 binding sites using ChIP-Seq, potentially utilizing the biotin-conjugated antibody for enhanced sensitivity.

• Integrate with Sp1 ChIP-Seq data to identify co-regulated genes.

• Focus analysis on cell cycle-related gene promoters and enhancers.

3. Gene Expression Analysis Following PTTG1 Modulation:

• Implement PTTG1 knockdown (siRNA/shRNA) or overexpression experiments.

• Analyze expression changes in G1/S transition regulators, particularly those identified as potential PTTG1 targets like cyclin D3 .

• Perform rescue experiments to determine which PTTG1 domains are essential for its effects on cell cycle progression.

4. Protein-Protein Interaction Studies:

• Expand on the known PTTG1-Sp1 interaction using techniques such as proximity ligation assays, FRET, or BiFC to visualize these interactions in living cells.

• Perform immunoprecipitation with the PTTG1 antibody followed by mass spectrometry to identify novel interaction partners in a cell cycle-dependent manner.

5. Functional Readouts:

• Measure proliferation rates, cell cycle distribution, and S-phase entry following PTTG1 manipulation.

• Correlate these phenotypes with molecular changes in the expression of cell cycle regulators.

What are common technical challenges when working with PTTG1 Antibody, Biotin conjugated and how can they be addressed?

Researchers working with PTTG1 Antibody, Biotin conjugated may encounter several technical challenges that require specific troubleshooting approaches:

1. Non-specific Binding:

• Challenge: Background signal in immunoassays due to non-specific binding.

• Solution: Optimize blocking conditions using 3-5% BSA or 5% non-fat dry milk. For biotin-conjugated antibodies, include an avidin/biotin blocking step to reduce endogenous biotin-related background.

2. Signal Variability:

• Challenge: Inconsistent signal strength between experiments.

• Solution: Prepare single-use aliquots to avoid freeze-thaw cycles . Standardize incubation times and temperatures. For quantitative applications, include calibration standards in each experiment.

3. Cross-Reactivity:

• Challenge: Potential cross-reactivity with related proteins.

• Solution: Include specific positive controls (recombinant PTTG1) and negative controls (PTTG1 knockdown samples). Validate specificity through peptide competition assays.

4. Detection Sensitivity:

• Challenge: Insufficient signal when detecting low-abundance endogenous PTTG1.

• Solution: Leverage the biotin conjugation by using high-sensitivity streptavidin detection systems. Consider tyramide signal amplification (TSA) for immunohistochemistry applications.

5. Epitope Masking:

• Challenge: Protein-protein interactions (such as with Sp1 ) may mask the epitope.

• Solution: Test different fixation and antigen retrieval methods. For co-immunoprecipitation experiments, consider mild detergent conditions that preserve protein complexes while allowing antibody access.

How can researchers validate the specificity of PTTG1 Antibody in their experimental systems?

Thorough validation of antibody specificity is critical for generating reliable research results. For PTTG1 Antibody, Biotin conjugated, consider these validation approaches:

1. Genetic Controls:

• Utilize PTTG1 knockout or knockdown models (CRISPR/Cas9, siRNA) as negative controls.

• Employ PTTG1 overexpression systems as positive controls.

• Compare signal between wild-type and genetically modified samples.

2. Peptide Competition Assay:

• Pre-incubate the antibody with excess immunizing peptide (Recombinant Human Securin protein, aa 2-202) .

• A specific antibody will show decreased or absent signal when the immunizing peptide blocks binding sites.

3. Multiple Antibody Validation:

• Compare results with other antibodies targeting different PTTG1 epitopes.

• Consistent results across antibodies increase confidence in specificity.

4. Correlation with mRNA Expression:

• Correlate protein detection levels with PTTG1 mRNA levels (qRT-PCR or RNA-seq).

• Consistent correlation supports antibody specificity.

5. Mass Spectrometry Verification:

• Perform immunoprecipitation using the PTTG1 antibody followed by mass spectrometry analysis.

• Confirm that PTTG1 is among the enriched proteins.

6. Expected Molecular Weight Confirmation:

• Verify that the detected band in Western blots corresponds to the expected molecular weight of PTTG1 (approximately 22-24 kDa).

• Check for known post-translational modifications that might affect migration patterns.

What experimental design considerations are important when investigating PTTG1's role in tumor progression?

When designing experiments to study PTTG1's role in tumor progression, researchers should consider:

1. Cell and Tissue Model Selection:

• Include both normal and malignant cell lines/tissues to establish baseline expression differences.

• Consider multiple cancer types, as PTTG1 has been implicated in various cancers including pituitary tumors, thyroid cancer, breast cancer, and colon cancer .

• Select models representing different stages of tumor progression to capture temporal changes in PTTG1 function.

2. Multi-level Analysis Approach:

• Combine protein expression analysis (using the biotin-conjugated PTTG1 antibody) with functional assays.

• Correlate PTTG1 levels with proliferation rates, invasion capacity, and angiogenesis markers.

• Use both in vitro and in vivo models to validate findings.

3. PTTG1 Manipulation Strategies:

• Implement both loss-of-function (siRNA, shRNA, CRISPR/Cas9) and gain-of-function (overexpression) approaches.

• Consider inducible systems to study temporal effects of PTTG1 modulation.

• Design domain-specific mutants to dissect the contribution of different PTTG1 functions (e.g., Sp1 interaction domain ).

4. Pathway Integration Analysis:

• Investigate PTTG1's relationship with known oncogenic pathways.

• Focus on the PTTG1-Sp1 axis and downstream effects on cell cycle regulators like cyclin D3 .

• Consider potential crosstalk with other transcription factors and signaling pathways.

5. Translational Relevance:

• Correlate experimental findings with patient data, as PTTG1 overexpression has been associated with tumor development, size, malignancy, and invasiveness .

• Consider potential therapeutic implications, as PTTG1 knockdown has shown protective effects against tumor formation in mouse models .

By implementing these experimental design considerations, researchers can generate comprehensive insights into PTTG1's multifaceted roles in tumor progression, potentially identifying new therapeutic targets or prognostic markers.

How might PTTG1 Antibody be utilized in identifying novel transcriptional targets beyond known interactions with Sp1?

Research has established that PTTG1 functions as a global transcription factor that interacts with Sp1 to regulate genes involved in cell cycle progression . To identify novel transcriptional targets, researchers can employ the following approaches using PTTG1 Antibody, Biotin conjugated:

1. ChIP-Seq/ChIP-on-Chip Analysis:

• Perform genome-wide ChIP-Seq using the biotin-conjugated PTTG1 antibody to identify all potential binding sites across the genome.

• Previous ChIP-on-Chip analysis identified 746 gene promoters as potential PTTG1 binding sites (P<0.001) , suggesting a global transcriptional effect.

• Analyze enriched motifs in PTTG1-bound regions to identify potential co-factors beyond Sp1.

2. Integrative Multi-omics Approach:

• Combine ChIP-Seq data with RNA-Seq analysis following PTTG1 modulation to correlate binding with transcriptional outcomes.

• Integrate with proteomics data to identify post-transcriptional regulatory mechanisms.

• Use systems biology approaches to construct comprehensive gene regulatory networks centered on PTTG1.

3. Sequential ChIP (Re-ChIP) with Novel Partners:

• Extend the sequential ChIP approach demonstrated with Sp1 to identify co-occupancy with other transcription factors.

• Select candidate factors based on motif analysis of PTTG1-bound regions.

• Focus on transcription factors involved in cancer progression and cell cycle regulation.

4. Targeted Promoter Analysis:

• Based on ChIP-Seq results, select novel promoters for detailed analysis similar to the cyclin D3 promoter study .

• Perform promoter mutagenesis to identify specific binding elements.

• Use reporter gene assays to validate functional relevance of PTTG1 binding.

This systematic approach would significantly expand our understanding of PTTG1's role as a transcriptional regulator, potentially revealing new therapeutic targets in PTTG1-overexpressing cancers.

What are the implications of PTTG1 biomarker studies for personalized cancer treatment approaches?

PTTG1 has emerged as a significant biomarker with implications for personalized cancer treatment approaches:

1. Prognostic Value:

• PTTG1 has been suggested as a prognostic marker for differentiated thyroid cancer, lymph node invasion, breast cancer recurrence, and colon cancer invasiveness and vascularity .

• Enhanced PTTG1 tumor abundance correlates with tumor development, size, and malignancy .

• The biotin-conjugated antibody could be utilized in tissue microarray studies to validate these associations across larger patient cohorts.

2. Patient Stratification:

• PTTG1 expression levels could potentially stratify patients for different treatment approaches.

• Using immunohistochemistry with the biotin-conjugated antibody, researchers could establish expression thresholds that correlate with treatment responses.

• This stratification could inform decisions regarding aggressive versus conservative treatment approaches.

3. Therapeutic Target Development:

• Studies using PTTG1-knockout mice have shown protection against tumor formation , suggesting PTTG1 knockdown as a potential therapeutic strategy.

• The antibody could be used to screen for compounds that modulate PTTG1 expression or activity.

• Monitoring PTTG1 levels during treatment could serve as a pharmacodynamic marker.

4. Monitoring Treatment Response:

• Serial measurement of PTTG1 expression in liquid biopsies or accessible tissues could monitor treatment efficacy.

• Changes in PTTG1 levels or localization might predict response or resistance to specific therapies.

• The biotin-conjugated antibody could be adapted for such monitoring assays.

5. Combination Therapy Rationale:

• Understanding PTTG1's interaction with Sp1 and effect on cell cycle regulators like cyclin D3 could inform rational combination therapies.

• For example, combining PTTG1-targeted approaches with cell cycle inhibitors might provide synergistic effects.

Translating these findings into clinical practice would require rigorous validation studies and standardized protocols for PTTG1 detection, where the biotin-conjugated antibody could play a central role due to its enhanced sensitivity through biotin-streptavidin detection systems.

What antibody dilution ranges are optimal for different applications of PTTG1 Antibody, Biotin conjugated?

Based on the properties of PTTG1 Antibody, Biotin conjugated and related antibodies in the product line, the following dilution ranges are recommended:

*Note: While the product is primarily recommended for ELISA , these dilutions are suggested starting points for optimization if adapting the antibody to other applications, based on similar antibodies in the product line.

What is the comparative effectiveness of PTTG1 detection methods in different experimental contexts?

The following table compares various PTTG1 detection methods and their effectiveness in different experimental contexts:

*Note: While the PTTG1 Antibody, Biotin conjugated is primarily recommended for ELISA , this comparative table provides guidance for researchers considering various detection methods based on their experimental needs.

What key experimental findings have been reported regarding PTTG1's role in cell cycle regulation?

The following table summarizes key experimental findings regarding PTTG1's role in cell cycle regulation:

These findings collectively establish PTTG1 as a critical regulator of cell cycle progression, particularly at the G1/S transition, through its interaction with transcription factors like Sp1 and subsequent regulation of cell cycle genes such as cyclin D3.

What are promising directions for expanding PTTG1 antibody applications in cancer research?

Several promising research directions could expand the utility of PTTG1 antibody in cancer research:

1. Liquid Biopsy Development:

• Adapt the biotin-conjugated PTTG1 antibody for highly sensitive detection of PTTG1 in circulation.

• Develop assays to detect PTTG1 in extracellular vesicles as potential non-invasive cancer biomarkers.

• Combine with digital ELISA technologies (e.g., Simoa) for single-molecule detection sensitivity.

2. Multiplex Imaging:

• Integrate the biotin-conjugated antibody into multiplex imaging platforms.

• Combine with other cancer biomarkers to create comprehensive tumor classification systems.

• Apply to spatial transcriptomics approaches to correlate PTTG1 protein localization with gene expression patterns.

3. Therapeutic Monitoring:

• Develop standardized PTTG1 quantification protocols for monitoring treatment response.

• Create companion diagnostic assays for emerging therapies targeting cell cycle checkpoints.

• Establish PTTG1 expression thresholds that correlate with treatment outcomes.

4. Drug Discovery Applications:

• Utilize the antibody in high-throughput screening systems to identify compounds that modulate PTTG1 expression or activity.

• Develop cell-based assays with PTTG1 antibody readouts for drug efficacy testing.

• Create PTTG1 reporter systems for real-time monitoring of drug effects.

5. Combinatorial Biomarker Approaches:

• Integrate PTTG1 detection with other cell cycle regulators and transcription factors.

• Develop multiplexed assays that simultaneously detect PTTG1, Sp1, and downstream targets like cyclin D3 .

• Create algorithms that incorporate PTTG1 expression patterns into comprehensive prognostic models.

These approaches would significantly expand the utility of PTTG1 antibodies beyond basic research applications, potentially creating new diagnostic and therapeutic monitoring tools for clinical oncology.

How might emerging technologies enhance the utility of biotin-conjugated antibodies like PTTG1 in molecular research?

Emerging technologies offer exciting opportunities to enhance the utility of biotin-conjugated antibodies like PTTG1 in molecular research:

1. Advanced Imaging Technologies:

• Super-resolution microscopy: Combining biotin-conjugated PTTG1 antibody with streptavidin-conjugated quantum dots or organic fluorophores for nanoscale imaging of PTTG1 localization.

• Expansion microscopy: Physical expansion of specimens after PTTG1 antibody labeling for enhanced spatial resolution of protein complexes.

• Lattice light-sheet microscopy: For capturing dynamic PTTG1 movements in living cells with minimal phototoxicity.

2. Single-Cell Analysis Platforms:

• Mass cytometry (CyTOF): Using metal-tagged streptavidin for detection of biotin-conjugated PTTG1 antibody in high-dimensional single-cell analysis.

• Single-cell proteomics: Adapting biotin-conjugated antibodies for sensitive detection in microfluidic platforms.

• Spatial proteomics: Mapping PTTG1 distribution across tissue architectures at single-cell resolution.

3. Proximity-Based Technologies:

• Proximity ligation assay (PLA): For highly sensitive detection of PTTG1-protein interactions, such as with Sp1 .

• BioID or APEX2 proximity labeling: Potential fusion of biotin ligases to PTTG1 to map its proximal interactome.

• FRET-based biosensors: For monitoring PTTG1 interactions in real-time in living cells.

4. Multi-omics Integration:

• CITE-seq/REAP-seq: Combining transcriptome analysis with PTTG1 protein detection at single-cell level.

• Spatial transcriptomics with protein detection: Correlating PTTG1 protein localization with local gene expression profiles.

• Integrative computational frameworks: Developing algorithms that synthesize PTTG1 data across multiple platforms.

5. Advanced Biotechnology Approaches:

• DNA-barcoded antibodies: For highly multiplexed PTTG1 detection alongside hundreds of other proteins.

• Nanobody development: Creating smaller biotin-conjugated binding reagents against PTTG1 for improved tissue penetration.

• Aptamer alternatives: Developing nucleic acid aptamers as alternatives to antibodies for PTTG1 detection.