TP53I11 Antibody, HRP conjugated

What is TP53I11 and why is it important in cancer research?

TP53I11 (Tumor Protein P53 Inducible Protein 11), also known as PIG11, is a 177 amino acid tumor suppressor belonging to the p53-induced protein gene (PIG) family. This protein family encodes redox-controlling proteins involved in p53 tumor suppressor activity. TP53I11 plays a significant role in tumor suppression through promotion of cell apoptosis and is particularly associated with arsenic trioxide As(2)O(3)-induced apoptosis in certain cell lines. The gene encoding TP53I11 maps to human chromosome 11, which contains over 1,400 genes and comprises nearly 4% of the human genome .

Research importance derives from TP53I11's central role in regulating extracellular matrix (ECM)-independent survival, epithelial-mesenchymal transition (EMT), and cell migration processes that are critical for understanding cancer metastasis mechanisms .

What are the key differences between various types of TP53I11 antibodies available for research?

TP53I11 antibodies are available in multiple formats with distinct properties that determine their suitability for different experimental applications:

The choice between these antibodies depends on the experimental design requirements, with HRP-conjugated versions particularly advantageous for direct detection systems that eliminate the need for secondary antibody incubation steps .

How does HRP conjugation affect antibody performance in TP53I11 detection?

HRP (horseradish peroxidase) conjugation provides direct enzymatic activity to primary antibodies targeting TP53I11, offering several methodological advantages:

• Streamlined detection protocols by eliminating secondary antibody incubation and washing steps, potentially reducing experimental time by 1-2 hours.

• Reduced background signal in some applications by minimizing non-specific binding associated with secondary antibodies.

• Enhanced sensitivity through direct enzymatic amplification of signal at the target epitope.

• Compatibility with multiple substrates (TMB, DAB, chemiluminescent reagents) allowing flexibility in detection methods.

What are the optimal conditions for using HRP-conjugated TP53I11 antibodies in Western blotting?

Optimizing Western blot detection of TP53I11 using HRP-conjugated antibodies requires careful consideration of several parameters:

• Sample preparation: Total cell lysates from cells expressing TP53I11 (such as MCF-7 or CEM cell lines) should be prepared under reducing conditions using RIPA buffer supplemented with protease inhibitors.

• Protein loading: 20-30 μg of total protein per lane generally provides detectable signals. For induced systems (e.g., after ionizing radiation treatment), comparison between treated (+) and untreated (-) samples is recommended.

• Dilution optimization: A working dilution of 1:5000 has been validated for many HRP-conjugated p53 family antibodies, but titration experiments (1:1000 to 1:10000) are recommended for each new lot or cell type.

• Membrane selection: PVDF membranes show superior performance compared to nitrocellulose for TP53I11 detection.

• Blocking conditions: 5% non-fat dry milk in TBST (0.1% Tween-20) for 1 hour at room temperature is generally effective.

• Incubation conditions: Overnight incubation at 4°C typically yields the best signal-to-noise ratio.

• Detection system: Enhanced chemiluminescence (ECL) systems provide the sensitivity required for detecting endogenous levels of TP53I11, which typically appears as a band at approximately 53 kDa.

For experimental validation, positive controls using cell lines with known TP53I11 expression (MCF-7, MDA-MB-231, A549) and negative controls using knockdown or knockout cell lines are strongly recommended .

How can researchers effectively use TP53I11 antibodies to study p53-dependent and p53-independent pathways?

Distinguishing between p53-dependent and p53-independent pathways involving TP53I11 requires carefully designed experimental approaches:

For p53-dependent pathway analysis:

• Create parallel experimental systems using isogenic cell lines with wild-type p53, mutant p53, and p53-null backgrounds.

• Employ stress induction protocols (e.g., DNA damage via 10 Gy ionizing radiation or 5-10 μM doxorubicin treatment) to activate p53.

• Monitor TP53I11 expression changes via Western blot or qRT-PCR in a time-dependent manner (0, 2, 6, 12, 24 hours post-treatment).

• Validate p53 dependency using p53 inhibitors (e.g., pifithrin-α) or siRNA-mediated knockdown.

• Perform chromatin immunoprecipitation (ChIP) to confirm direct p53 binding to the TP53I11 promoter region.

For p53-independent pathway analysis:

• Establish TP53I11 overexpression and knockdown systems in p53-null cell backgrounds.

• Analyze downstream effects on cellular processes like ECM-independent survival, EMT, and cell migration.

• Investigate TP53I11 interactions with AMPK signaling pathways through co-immunoprecipitation studies.

• Monitor effects on AKT/mTOR/p70S6K signaling components in both attached and detached culture conditions.

Recent research has shown that TP53I11 can regulate ECM-independent survival through a p53-independent mechanism involving the balance between AKT and AMPK activation pathways, suggesting broader roles for this protein beyond its initially characterized p53-responsive functions .

What considerations are important when using HRP-conjugated antibodies for detection of mutant versus wild-type p53-related proteins?

When distinguishing between mutant and wild-type p53-related proteins using HRP-conjugated antibodies, researchers should consider several critical factors:

• Antibody epitope selection: Choose antibodies targeting regions distinct from common mutation hotspots in p53 for detection of total p53 family proteins, or select mutation-specific antibodies for distinguishing variant forms.

• Expression level differences: Mutant p53 proteins often accumulate to higher levels than wild-type forms due to impaired degradation mechanisms. Adjust exposure times and antibody dilutions accordingly to prevent signal saturation.

• Cross-reactivity assessment: Validate antibody specificity using cell lines with defined p53 status (wild-type, null, and specific mutations) to confirm absence of cross-reactivity.

• Post-translational modifications: Consider that mutant and wild-type p53 proteins may exhibit different patterns of phosphorylation, acetylation, and other modifications that could affect antibody recognition.

• Conformation-dependent recognition: Some p53 antibodies recognize conformation-dependent epitopes that differ between mutant and wild-type proteins; HRP conjugation may potentially affect these recognition properties.

The TCR-like antibody P1C1TM provides an excellent example of distinguishing between mutant and wild-type p53 expressing cells by recognizing the p53125-134 peptide in complex with HLA-A24. This approach demonstrates how peptide-MHC complexes can serve as specific targets for immunotherapy against mutant p53 expressing tumors .

How does TP53I11 regulation of AMPK activation affect cellular metabolism in cancer cells?

TP53I11 plays a sophisticated role in metabolic regulation through modulating the AMPK pathway in a context-dependent manner:

In attached cancer cells:
Loss of TP53I11 promotes activation of the AKT/mTOR pathway, increases PGC-1α expression, and enhances oxidative phosphorylation (OXPHOS), creating a metabolic state that supports proliferation and growth.

In detached cancer cells (modeling metastatic conditions):
Loss of TP53I11 shifts toward promoting AMPK activation, which inhibits AKT/mTOR/p70S6K signaling, enabling cellular adaptation to ECM-detachment stress and promoting survival.

This metabolic plasticity regulation is critical for understanding how cancer cells adapt to changing environmental conditions during metastasis. The table below summarizes metabolic pathway changes in MCF10A cells with TP53I11 knockdown:

These findings suggest that targeting TP53I11 or its regulated pathways requires consideration of the cellular context, as the same molecular intervention could have opposing effects depending on the attachment status of cancer cells .

What are the most effective experimental approaches for studying TP53I11's role in extracellular matrix-independent survival?

Investigating TP53I11's functions in ECM-independent survival requires sophisticated experimental approaches:

• 3D culture systems and anoikis assays:

  • Ultra-low attachment plates coated with poly-HEMA

  • Forced suspension culture in methylcellulose

  • Hanging drop spheroid formation

  • Quantification via viability assays (CellTiter-Glo, LIVE/DEAD staining)

• Genetic manipulation strategies:

  • CRISPR/Cas9-mediated TP53I11 knockout

  • Inducible shRNA systems for temporal control of knockdown

  • Rescue experiments with wild-type and mutant TP53I11 constructs

• Signaling pathway analysis:

  • Phospho-specific antibodies to monitor AMPK (Thr172) and AKT (Ser473) activation

  • Specific pathway inhibitors: Compound C (AMPK inhibitor), MK-2206 (AKT inhibitor)

  • Time-course studies comparing attached versus detached states (0, 6, 12, 24, 48 hours)

• Metabolic profiling:

  • Seahorse XF analysis for oxygen consumption rate (OCR) measurement

  • FCCP-induced maximal respiration assessment

  • Metabolite quantification via LC-MS

• In vivo metastasis models:

  • Tail vein injection for experimental metastasis

  • Spontaneous metastasis models with primary tumor resection

  • Ex vivo lung colonization assays

The combination of these approaches allows comprehensive assessment of how TP53I11 regulates the balance between AMPK and AKT signaling pathways to adapt cells to changing ECM conditions during metastatic progression .

How can HRP-conjugated TP53I11 antibodies be used in multiplexed detection systems for studying p53 pathway activation?

Advanced multiplexed detection systems using HRP-conjugated TP53I11 antibodies provide powerful tools for simultaneously analyzing multiple components of the p53 pathway:

• Sequential multiplexed Western blotting:

  • Strip and reprobe membranes using harsh stripping buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 0.8% β-mercaptoethanol)

  • Use HRP-conjugated antibodies with different substrates producing distinct colorimetric reactions

  • Carefully validate complete stripping between detection cycles

• Multiplex immunohistochemistry/immunofluorescence:

  • Tyramide signal amplification (TSA) with HRP-conjugated antibodies

  • Sequential antibody staining with microwave treatment for antibody removal

  • Spectral unmixing to separate overlapping fluorescent signals

• Bead-based multiplex assays:

  • Conjugation of capture antibodies to distinctly coded beads

  • Detection using HRP-conjugated detection antibodies

  • Fluorescent readout via flow cytometry or dedicated bead analyzers

• Protein array technologies:

  • Reverse phase protein arrays (RPPA) with HRP detection

  • Antibody arrays for detecting multiple p53 pathway components

  • Quantitative analysis using standard curves

• Single-cell multiplexed detection:

  • Mass cytometry (CyTOF) with metal-conjugated antibodies

  • Imaging mass cytometry for spatial context

  • Digital spatial profiling with oligo-barcoded antibodies

These technologies enable researchers to simultaneously monitor TP53I11 expression alongside other p53 pathway components (MDM2, p21, BAX, PUMA) and signaling intermediates (phospho-AMPK, phospho-AKT) to build comprehensive profiles of pathway activation states under various experimental conditions .

What are common issues encountered when using HRP-conjugated antibodies for TP53I11 detection and how can they be resolved?

Researchers frequently encounter several challenges when using HRP-conjugated antibodies for TP53I11 detection:

For particularly challenging samples, consider alternative detection strategies such as biotin-streptavidin amplification systems or tyramide signal amplification to enhance sensitivity while maintaining specificity .

How can researchers validate the specificity of TP53I11 antibodies in various experimental systems?

Rigorous validation of TP53I11 antibody specificity is essential for generating reliable research data:

• Genetic validation approaches:

  • CRISPR/Cas9 knockout of TP53I11 in relevant cell lines

  • siRNA/shRNA knockdown with multiple constructs targeting different regions

  • Overexpression of tagged TP53I11 for parallel detection with tag-specific antibodies

  • Use of cells from TP53I11 knockout animal models where available

• Biochemical validation methods:

  • Pre-absorption with immunizing peptide to confirm specific binding

  • Dot blot analysis with recombinant TP53I11 protein and unrelated proteins

  • Comparison of multiple antibodies targeting different epitopes

  • Mass spectrometry confirmation of immunoprecipitated proteins

• Application-specific validations:

  • For Western blotting: Observe band at expected molecular weight (~53 kDa)

  • For IHC/IF: Compare staining patterns with mRNA expression data

  • For ELISA: Generate standard curves with recombinant protein

  • For IP-based applications: Confirm enrichment by Western blot

• Cross-species reactivity assessment:

  • Test antibodies against lysates from multiple species (human, mouse, rat)

  • Compare observed patterns with predicted cross-reactivity

  • Validate knockdown/knockout effects across species-specific cell lines

The gold standard validation combines multiple approaches, particularly genetic manipulation of the target with antibody detection to confirm specificity under experimental conditions relevant to the research question .

What quality control measures should be implemented when using HRP-conjugated antibodies in long-term research projects?

Maintaining consistent and reliable results with HRP-conjugated antibodies over extended research projects requires systematic quality control procedures:

• Initial antibody characterization:

  • Determine optimal working dilution range through titration experiments

  • Establish limits of detection using standard curves with recombinant protein

  • Document batch/lot information and initial performance metrics

  • Aliquot and store antibodies according to manufacturer recommendations

• Regular performance monitoring:

  • Include consistent positive and negative controls in each experiment

  • Track signal-to-noise ratios and detection sensitivity over time

  • Monitor HRP activity using standardized substrates

  • Document exposure times and image acquisition settings

• Storage and stability testing:

  • Compare freshly thawed aliquots against previously used material

  • Test antibody performance after different storage durations

  • Evaluate freeze-thaw stability if relevant to laboratory practices

  • Consider accelerated stability testing for critical applications

• Standardized protocols and documentation:

  • Maintain detailed protocols with all critical parameters

  • Document any protocol deviations and their effects

  • Use laboratory information management systems (LIMS) when available

  • Implement electronic laboratory notebooks for consistent documentation

• Reference standard development:

  • Create internal reference standards from well-characterized samples

  • Maintain a standard curve of recombinant TP53I11 protein

  • Consider developing stable cell lines with defined TP53I11 expression

  • Archive representative images/data from successful experiments

• Periodic revalidation:

  • Repeat specificity testing with each new antibody lot

  • Reconfirm applications and dilutions annually

  • Verify continued reactivity with target after protocol modifications

  • Compare new lots against reference standards before depleting old stock

Implementation of these measures ensures data reproducibility and facilitates troubleshooting when unexpected results occur during long-term research projects .

How are TP53I11 antibodies being utilized in developing targeted cancer therapies?

TP53I11 antibodies are finding novel applications in targeted cancer therapy development:

• Antibody-drug conjugates (ADCs): Research is exploring the potential of conjugating cytotoxic agents to TP53I11-targeting antibodies for selective delivery to cancer cells with aberrant TP53I11 expression.

• TCR-like antibody approaches: Following the model of P1C1TM antibody that recognizes p53-derived peptide-MHC complexes, similar approaches targeting TP53I11-derived peptides presented by MHC molecules could enable selective targeting of cells with altered TP53I11 processing.

• Immunotherapy enhancement: TP53I11 antibodies may help identify tumors with dysregulated p53 pathway activation that might respond to specific immunotherapy approaches, particularly in combination with checkpoint inhibitors.

• Antibody-enabled drug screening: HRP-conjugated TP53I11 antibodies facilitate high-throughput screening assays to identify compounds that modulate TP53I11 expression or post-translational modifications.

The development of PNU-159682-P1C1TM drug conjugates that specifically inhibit growth of mutant p53 expressing cells both in vitro and in vivo demonstrates the potential of targeting p53 pathway components like TP53I11 for cancer therapy .

What recent technological advances are improving the sensitivity and specificity of HRP-conjugated antibody detection systems?

Recent technological innovations have significantly enhanced the performance of HRP-conjugated antibody detection systems:

• Enhanced enzymatic substrates:

  • Super-sensitive chemiluminescent substrates with femtogram detection limits

  • Extended dynamic range formulations for quantitative applications

  • Substrates with reduced background and improved signal stability

• Signal amplification technologies:

  • Tyramide signal amplification (TSA) providing 10-100× sensitivity enhancement

  • Poly-HRP systems with multiple enzyme molecules per antibody

  • Cascade enzyme amplification using coupled enzymatic reactions

• Microfluidic integration:

  • Automated microfluidic immunoassay platforms

  • Reduced sample and reagent requirements

  • Improved reaction kinetics through optimized fluid dynamics

• Digital detection methods:

  • Digital ELISA platforms with single-molecule detection capability

  • Digital imaging of enzyme-generated precipitates

  • Machine learning algorithms for improved signal discrimination

• Novel conjugation chemistries:

  • Site-specific conjugation to preserve antibody functionality

  • Controlled orientation of HRP molecules relative to binding sites

  • Reduced batch-to-batch variability through defined conjugation ratios

These advances collectively enable detection of TP53I11 at physiologically relevant concentrations in complex biological samples with improved reliability and quantitative accuracy .

How can researchers integrate TP53I11 antibody-based detection with functional genomics approaches to better understand p53 pathway dysregulation in cancer?

Integrating TP53I11 antibody-based detection with functional genomics creates powerful research paradigms:

• CRISPR screening with antibody-based readouts:

  • Genome-wide or focused CRISPR libraries targeting p53 pathway components

  • HRP-conjugated TP53I11 antibodies for high-throughput detection

  • Automated image analysis to quantify expression changes

  • Identification of novel regulators of TP53I11 expression and localization

• Single-cell multi-omics integration:

  • Combine single-cell proteomics using antibody-based detection

  • Parallel single-cell transcriptomics and/or epigenomics

  • Computational integration to identify correlations between TP53I11 protein levels and transcriptional programs

• Spatial transcriptomics with protein validation:

  • Spatial mapping of TP53I11 mRNA expression in tissue sections

  • Validation and correlation with protein expression using HRP-conjugated antibodies

  • Integration with cell type markers and microenvironmental features

• Proteogenomic correlation studies:

  • Systematic correlation between TP53I11 genetic alterations and protein expression

  • Impact of p53 pathway mutations on TP53I11 protein levels and modifications

  • Association with broader proteome remodeling in cancer progression

• High-content phenotypic screening:

  • Multiplex detection of TP53I11 alongside cell morphology and viability

  • Correlation with genetic perturbations or drug treatments

  • Machine learning classification of complex cellular phenotypes

By combining these approaches, researchers can build comprehensive models of how TP53I11 functions within the broader p53 pathway and identify novel therapeutic vulnerabilities in cancers with p53 pathway dysregulation .