TP53I3 Antibody

What is TP53I3 and why is it significant in cancer research?

TP53I3 is a quinone oxidoreductase that participates in the generation of reactive oxygen species (ROS) and has NADPH-dependent naphtoquinone reductase activity, with a preference for 1,2-naphtoquinone over 1,4-naphtoquinone . It is primarily localized to the cytoplasm and is induced in primary, non-transformed, and transformed cell cultures after exposure to genotoxic agents . As a p53-inducible gene, it plays a role in p53-mediated apoptosis pathways, making it valuable for studying cellular responses to stress, DNA damage, and cancer progression.

TP53I3 microsatellite polymorphism has been associated with differential susceptibility to cancer , suggesting its potential as a biomarker or therapeutic target. Understanding TP53I3 expression and function can provide insights into the p53 pathway, which is central to tumor suppression mechanisms.

What types of TP53I3 antibodies are available for research applications?

Several types of TP53I3 antibodies are available for research purposes, including:

These antibodies vary in their specificity, reactivity with different species (primarily human, though some cross-react with mouse and rat), and optimal applications. The diversity of available antibodies allows researchers to select the most appropriate tool based on their specific experimental requirements.

What are the recommended applications for TP53I3 antibodies?

TP53I3 antibodies can be used in multiple research applications, with validated results in the following techniques:

• Western Blotting (WB): For detecting TP53I3 protein expression levels in cell lysates

• Immunohistochemistry (IHC): For examining TP53I3 expression patterns in tissue sections

• Immunocytochemistry (ICC): For visualizing TP53I3 in cultured cells

• Immunofluorescence (IF): For studying the subcellular localization of TP53I3

• ELISA: For quantitative detection of TP53I3 in solution

• Immunoprecipitation (IP): For isolating TP53I3 protein complexes

The choice of application should be guided by the specific research question, the sensitivity required, and the nature of the samples being analyzed.

How should TP53I3 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of TP53I3 antibodies are crucial for maintaining their activity:

• Storage temperature: Store at -20°C for long-term preservation (up to 12 months)

• Short-term storage: Can be kept at 4°C for up to 1 month

• Avoid freeze-thaw cycles: Repeated freezing and thawing can degrade antibody quality

• Formulation: Typically supplied in PBS with preservatives like 0.02% sodium azide and stabilizers such as 50% glycerol at pH 7.3

• Aliquoting: For antibodies without glycerol, aliquoting is recommended to avoid repeated freeze-thaw cycles

Following manufacturer-specific guidelines is essential as storage conditions may vary depending on the specific antibody formulation and conjugation status.

What are the optimal dilutions for different applications of TP53I3 antibodies?

Optimal dilutions vary by application and specific antibody. Based on the search results, here are recommended starting dilutions:

Always titrate the antibody in your specific experimental system to determine optimal concentration. Different antibody batches may require slight adjustments to these recommendations.

How can I validate the specificity of a TP53I3 antibody?

Validating antibody specificity is crucial for reliable results. Here are methodological approaches:

• Genetic validation: Compare detection in wild-type versus TP53I3 knockdown/knockout samples

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

• Multiple antibodies: Use antibodies targeting different epitopes of TP53I3 to confirm results

• Molecular weight verification: Confirm observed band matches the expected size (approximately 36-37 kDa for TP53I3)

• Positive controls: Use cell lines known to express TP53I3 (e.g., A549 lung carcinoma cells)

• Cross-reactivity assessment: Test on samples from different species if cross-reactivity is claimed

• Immunoprecipitation-Mass spectrometry: Confirm the identity of the immunoprecipitated protein

Enhanced validation methods, such as orthogonal RNAseq and independent validation, provide additional confidence in antibody specificity .

What controls should I include when using TP53I3 antibodies in experimental procedures?

Proper controls are essential for interpreting results with TP53I3 antibodies:

For Western Blotting:

• Positive control: A431 or HEK-293 cells (known to express TP53I3)

• Loading control: Housekeeping proteins like GAPDH or β-actin

• Negative control: TP53I3 knockdown/knockout samples

• Molecular weight marker: To confirm band size matches expected 36 kDa

For Immunohistochemistry:

• Positive tissue controls: Human prostate, gliomas, endometrial, or ovary tumor tissues

• Negative control: Omit primary antibody incubation

• Isotype control: Non-specific antibody of same isotype

• Antigen retrieval comparison: Test both TE buffer pH 9.0 and citrate buffer pH 6.0

For Immunofluorescence:

• Positive control: HepG2 cells

• Counterstain: Nuclear marker (DAPI/Hoechst) to visualize cell context

• Negative control: Primary antibody omission

Including appropriate controls ensures reliable interpretation and troubleshooting of unexpected results.

What are key considerations when selecting between polyclonal and monoclonal TP53I3 antibodies?

The choice between polyclonal and monoclonal TP53I3 antibodies depends on your research needs:

Polyclonal TP53I3 Antibodies:

• Recognize multiple epitopes, potentially increasing detection sensitivity

• Ideal for proteins with low expression levels

• May show higher batch-to-batch variation

• Examples: Rabbit polyclonal antibodies targeting full-length TP53I3 or specific regions

Monoclonal TP53I3 Antibodies:

• Target single epitope, offering higher specificity

• More consistent between batches

• Better for applications requiring high reproducibility

• Examples: Mouse monoclonal antibody clone P1C9AT

Consider these factors:

• Application requirements: Some techniques benefit from signal amplification (polyclonal) while others require high specificity (monoclonal)

• Sample type: Native vs. denatured proteins may expose different epitopes

• Detection system: Some secondary antibodies work better with certain host species

• Research goal: Screening vs. quantitative analysis

For initial characterization, using both types of antibodies targeting different epitopes provides validation through complementary approaches.

How can TP53I3 antibodies be used to study p53-dependent apoptosis pathways?

TP53I3 antibodies offer valuable tools for investigating p53-dependent apoptosis through these methodological approaches:

• Induction kinetics: Use Western blotting with TP53I3 antibodies to monitor protein expression following p53 activation by genotoxic agents. This reveals the temporal relationship between p53 activation and TP53I3 induction.

• Subcellular localization: Employ immunofluorescence with TP53I3 antibodies to track protein localization changes during apoptosis progression, particularly in relation to mitochondria and ROS generation sites.

• Protein interactions: Utilize co-immunoprecipitation with TP53I3 antibodies to identify binding partners in the apoptotic cascade. This technique revealed historical interactions between p53 and viral proteins like SV40 T antigen and adenovirus E1b protein .

• Conformational changes: Specific antibodies can detect epitope changes during complex formation, similar to how PAb246 epitope on p53 was found to be unstable without bound SV40 T antigen . This approach could reveal functional states of TP53I3.

• Comparative analysis: Combine TP53I3 detection with other p53-regulated pro-apoptotic proteins to establish pathway hierarchies and dependencies.

These approaches can elucidate TP53I3's role in ROS generation and subsequent apoptotic events within the p53-mediated cell death pathway.

How do TP53I3 expression patterns differ between normal and transformed cell lines?

Understanding TP53I3 expression differences between normal and transformed cells provides insights into cancer biology:

• Baseline expression: TP53I3 is widely expressed in many tissue types but with variable levels . Research methodologies to characterize these differences include:

  • Western blot quantification normalized to housekeeping proteins

  • qRT-PCR to measure mRNA levels

  • Immunohistochemistry to assess tissue-specific patterns

• Induction patterns: TP53I3 is induced in primary, non-transformed, and transformed cell cultures after exposure to genotoxic agents . Experimental approaches to study this include:

  • Time-course analysis following genotoxic agent exposure

  • Comparison between isogenic cell lines with different p53 status

  • Dose-response studies with various DNA-damaging agents

• p53-dependence: Since TP53I3 is p53-inducible, its expression correlates with p53 status. Cells with mutant p53 may show altered TP53I3 expression. Research methods include:

  • Parallel detection of p53 and TP53I3 using specific antibodies

  • Correlation analysis of p53 mutation status with TP53I3 levels

  • p53 knockdown/knockout experiments to confirm dependency

• Clinical correlations: TP53I3 microsatellite polymorphism is associated with differential susceptibility to cancer , suggesting genetic variation affects function. Approaches include:

  • Genotype-phenotype correlation studies

  • Cancer tissue microarray analysis of TP53I3 expression

  • Survival analysis based on TP53I3 expression patterns

These methodological approaches can help distinguish normal biological variation from pathological alterations in TP53I3 expression.

What techniques are most effective for detecting TP53I3 in different subcellular compartments?

Since TP53I3 localization is functionally relevant to its role in ROS generation, these techniques are most effective for studying its subcellular distribution:

• Subcellular fractionation with Western blotting:

  • Methodology: Separate cellular components (cytoplasm, mitochondria, nucleus) using differential centrifugation

  • Detection: Probe fractions with TP53I3 antibodies (1:500-1:2000 dilution)

  • Controls: Include compartment-specific markers (e.g., GAPDH for cytoplasm, VDAC for mitochondria, Lamin for nucleus)

• High-resolution immunofluorescence microscopy:

  • Methodology: Fix cells with 4% paraformaldehyde, permeabilize, and incubate with TP53I3 antibodies (1:20-1:200 dilution)

  • Co-localization: Use organelle-specific markers and calculate Pearson's correlation coefficient

  • Advanced technique: Super-resolution microscopy (STED, STORM) for precise localization beyond diffraction limit

• Proximity ligation assay (PLA):

  • Methodology: Detect interactions between TP53I3 and compartment-specific proteins when they're within 40 nm

  • Advantage: Visualizes protein-protein interactions in situ with high sensitivity

  • Controls: Include positive interactions and random protein pairs as negative controls

• Immunoelectron microscopy:

  • Methodology: Label ultrathin sections with TP53I3 antibodies and gold-conjugated secondary antibodies

  • Advantage: Nanometer-scale resolution of protein localization

  • Analysis: Quantify gold particle distribution across cellular compartments

Since TP53I3 is primarily cytoplasmic , these techniques can reveal redistribution under stress conditions and provide insights into its functional interactions.

What are the methodological considerations for studying TP53I3's role in ROS generation?

Investigating TP53I3's role in reactive oxygen species (ROS) generation requires specific methodological approaches:

• Enzymatic activity assays:

  • NADPH-dependent reduction of quinones can be measured spectrophotometrically

  • Monitor preference for 1,2-naphtoquinone over 1,4-naphtoquinone

  • Measure NADPH consumption rate as indicator of enzymatic activity

• ROS detection methods:

  • Fluorescent probes: DCF-DA (general ROS), MitoSOX (mitochondrial superoxide)

  • Chemiluminescence assays: Lucigenin or luminol-based detection

  • EPR spectroscopy for specific radical identification

  • Flow cytometry for quantifying ROS levels in cell populations

• Genetic manipulation approaches:

  • TP53I3 overexpression: Observe dose-dependent effects on ROS production

  • CRISPR-Cas9 knockout: Determine baseline contribution to cellular ROS

  • Site-directed mutagenesis: Target catalytic residues to link enzymatic activity to ROS production

• Correlation with oxidative damage:

  • Measure 8-oxo-dG levels (DNA oxidation)

  • Detect lipid peroxidation products (MDA, 4-HNE)

  • Assess protein carbonylation as marker of oxidative protein damage

• Subcellular localization of ROS production:

  • Compartment-specific ROS probes

  • Co-localization of TP53I3 with sites of ROS generation

  • Mitochondrial function assessment (membrane potential, respiration)

These methodological approaches can establish the direct contribution of TP53I3 to cellular ROS levels and distinguish it from other sources of oxidative stress in experimental systems.

Why might I see multiple bands when using TP53I3 antibodies in Western blot?

Multiple bands in TP53I3 Western blots can occur for several reasons, each requiring specific troubleshooting approaches:

• Protein isoforms: Up to two different isoforms have been reported for TP53I3 . Verify if the observed bands match expected molecular weights.

• Post-translational modifications: Modifications can alter protein migration. Check if treatments affecting phosphorylation or other modifications change the band pattern.

• Degradation products: Incomplete protease inhibition during sample preparation. Verify by:

  • Using fresh samples

  • Adding additional protease inhibitors

  • Comparing different lysis protocols

• Non-specific binding: Cross-reactivity with structurally similar proteins. Address by:

  • Increasing antibody dilution (1:2000 instead of 1:500)

  • Longer blocking steps

  • Using more stringent washing conditions

  • Peptide competition assays to identify specific bands

• Protein complexes: Incomplete sample denaturation. Optimize by:

  • Increasing SDS concentration

  • Longer boiling time

  • Adding reducing agents

  • Using urea-based lysis buffers for resistant complexes

• Observed vs. calculated weight discrepancy: TP53I3 has a calculated molecular weight of 36 kDa but may be observed at 37 kDa or even 72 kDa . This could reflect:

  • Post-translational modifications

  • Dimerization

  • Anomalous migration due to protein structure

Validate bands by comparing multiple antibodies targeting different epitopes of TP53I3 to identify which bands represent the target protein.

How can I optimize immunohistochemical detection of TP53I3 in tissue samples?

Optimizing immunohistochemical detection of TP53I3 requires systematic approach to several variables:

• Antigen retrieval optimization:

  • Test both TE buffer pH 9.0 and citrate buffer pH 6.0

  • Compare heat-induced (pressure cooker, microwave) vs. enzymatic methods

  • Optimize retrieval duration (10-30 minutes)

• Antibody selection and dilution:

  • Test multiple antibodies against different epitopes

  • Perform titration series (1:50 - 1:500)

  • Consider overnight incubation at 4°C vs. shorter incubations at room temperature

• Detection system optimization:

  • Compare polymer-based vs. avidin-biotin systems

  • Evaluate different chromogens (DAB, AEC) for optimal signal-to-noise ratio

  • Test signal amplification methods for low-abundance targets

• Blocking optimization:

  • Increase blocking duration (1-2 hours)

  • Test different blocking agents (normal serum, BSA, commercial blockers)

  • Include avidin/biotin blocking for biotin-based detection systems

• Controls:

  • Positive tissue controls: Human prostate cancer, gliomas, endometrial cancer, ovary tumor tissues

  • Negative controls: Primary antibody omission and isotype controls

  • Competing peptide controls to verify specificity

• Counterstaining and mounting:

  • Optimize hematoxylin intensity to maintain nuclear detail without obscuring specific staining

  • Select appropriate mounting media for long-term preservation

When analyzing results, consider that TP53I3 expression may correlate with p53 status in the tissue, and staining patterns might differ between normal and neoplastic samples.

How do I interpret changes in TP53I3 expression following genotoxic treatments?

Interpreting TP53I3 expression changes after genotoxic treatments requires consideration of multiple factors:

• Temporal dynamics:

  • Early expression (2-6 hours): Direct p53-mediated transcriptional activation

  • Later expression (12-24 hours): May involve secondary regulatory mechanisms

  • Sustained vs. transient expression: Indicator of persistent vs. resolved DNA damage

• p53 dependency validation:

  • Compare wild-type vs. p53-null or mutant cells

  • Correlate with other p53 target genes (p21, MDM2, PUMA)

  • Consider p53 post-translational modifications that affect transcriptional activity

• Dose-response relationship:

  • Threshold effects: Minimum genotoxic dose required for TP53I3 induction

  • Saturation effects: Maximum expression levels at high doses

  • Biphasic responses: Potential adaptive vs. apoptotic signaling

• Cell type specificity:

  • Compare responses in different cell lineages

  • Consider baseline p53 pathway activity

  • Account for cell cycle distribution differences

• Correlation with functional outcomes:

  • ROS production: Using ROS-specific fluorescent probes

  • Apoptosis markers: Annexin V, caspase activation, PARP cleavage

  • Cell cycle analysis: PI staining, BrdU incorporation

• Methodological considerations:

  • Protein vs. mRNA dynamics: Transcriptional vs. post-transcriptional regulation

  • Single-cell vs. population analyses: Flow cytometry or immunofluorescence for heterogeneity assessment

  • Quantification methods: Densitometry normalization protocols for Western blots

These analytical approaches help distinguish between specific TP53I3 responses and generalized stress reactions, providing insights into the role of TP53I3 in the DNA damage response pathway.

What are the key methodological differences between various anti-p53 and anti-TP53I3 antibodies?

Understanding differences between p53 and TP53I3 antibodies is crucial for experimental design:

• Epitope recognition:

  • p53 antibodies: Many recognize terminal epitopes rather than the central mutation-containing domain

  • TP53I3 antibodies: Target various regions including full-length (AA 1-332), C-terminal regions, or specific sequences

• Conformational sensitivity:

  • Some p53 antibodies (like PAb246) are sensitive to conformational changes and recognize epitopes stable only when p53 is complexed with binding partners

  • Most TP53I3 antibodies detect denatured protein in Western blot, suggesting less conformational sensitivity

• Cross-reactivity profiles:

  • p53 antibodies: Some show exquisite species specificity (e.g., DO-1 binds human but not mouse p53 due to a single amino acid difference)

  • TP53I3 antibodies: May show cross-reactivity with human, mouse, and rat, depending on the specific antibody

• Application suitability:

  • Western Blot: Most antibodies for both proteins work well

  • Immunoprecipitation: Specific antibodies needed; affects complex detection

  • Native detection: Some antibodies only recognize folded vs. denatured forms

• Post-translational modification sensitivity:

  • Some p53 antibodies (like PAb421) recognize epitopes affected by phosphorylation

  • Limited information exists about TP53I3 antibodies' sensitivity to post-translational modifications

• Sensitivity to mutations:

  • p53 mutation-specific antibodies can distinguish between wild-type and mutant forms

  • TP53I3 mutation-specific antibodies are not widely available

This comparative analysis highlights the importance of selecting the appropriate antibody based on experimental goals and understanding the specific characteristics of each antibody for accurate data interpretation.