PAP10 Antibody

What is PABP and why is it an important research target?

PABP (Poly(A)-binding protein) is a crucial, well-conserved multifunctional protein that plays significant roles in translational initiation, mRNA biogenesis, and degradation. It is essential for cellular function as it interacts with polyadenylated mRNA, facilitating the recruitment of translation initiation factors and promoting efficient mRNA translation. This ensures protein synthesis machinery can access mRNA quickly, particularly during cellular stress or in response to signaling pathways that regulate gene expression. The human PABP gene maps to chromosome 8q22.2-q23 and encodes a 633 amino acid protein with four N-terminal RNA-binding domains and a C-terminus involved in protein-protein interactions .

What experimental applications is PABP Antibody (10E10) validated for?

PABP Antibody (10E10) is a mouse monoclonal IgG1 kappa light chain antibody that specifically detects Poly(A)-binding protein of human origin. It has been validated for multiple experimental techniques including western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), and immunohistochemistry. This versatility makes it a valuable tool for investigating PABP expression, localization, and interactions in various research contexts .

How does PAP expression differ across prostate cancer stages and why is this relevant?

PAP (Prostatic Acid Phosphatase) expression is highly restricted to prostate tissue and is present in approximately 95% of primary prostate tumors, making it an excellent candidate for targeted therapies. Expression patterns may vary across cancer stages, with implications for both diagnostic and therapeutic applications. Researchers investigating prostate cancer progression should consider monitoring PAP expression levels as a potential biomarker and therapeutic target, particularly in the context of developing immunotherapies for castrate-resistant prostate cancer (CRPC) .

What are the critical parameters for optimizing western blotting with PABP Antibody (10E10)?

When designing western blotting experiments with PABP Antibody (10E10), researchers should optimize several parameters:

• Sample preparation: Use lysis buffers with protease inhibitors to prevent PABP degradation

• Protein loading: 20-50 μg of total protein is typically sufficient for detecting PABP

• Gel percentage: 8-10% SDS-PAGE gels provide optimal resolution for the 633 aa PABP protein

• Transfer conditions: Semi-dry or wet transfer at appropriate voltage/time for complete transfer

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Antibody dilution: Start with 1:1000 dilution and optimize as needed

• Controls: Include positive controls (cells known to express PABP) and negative controls (PABP knockdown cells)

Researchers should expect a band at approximately 70 kDa corresponding to PABP. Non-specific binding can be addressed by adjusting antibody concentration, blocking conditions, and wash stringency .

What are the methodological considerations for studying PAP-specific T cell responses?

When investigating PAP-specific T cell responses for immunotherapy research, several methodological approaches are recommended:

• Flow cytometry with DextramerTM technology to detect and quantify PAP-specific CD8+ T cells in blood samples

• Cytokine secretion assays to measure IFNγ and TNFα production following stimulation

• Cytotoxicity assays to assess the killing capacity of PAP-stimulated T cells against target cells

• Analysis of memory T cell populations (effector vs. central memory) following vaccination

• In vitro stimulation of PBMCs with PAP peptides of different lengths to assess epitope recognition

• Comparative studies between wild-type and mutated PAP sequences to determine immunogenicity

These approaches provide comprehensive assessment of both quantity and functionality of PAP-specific immune responses, critical for evaluating potential immunotherapeutic interventions .

How should researchers select the appropriate PABP Antibody (10E10) conjugate for their experiments?

Selection of the appropriate PABP Antibody (10E10) conjugate depends on experimental requirements:

Researchers should consider signal amplification needs, multiplexing requirements, and detection system compatibility when selecting the appropriate format .

How can researchers utilize PABP Antibody (10E10) to investigate translational control during cellular stress?

Investigating translational control during cellular stress with PABP Antibody (10E10) requires several methodological approaches:

• Immunofluorescence time-course experiments to track PABP relocalization to stress granules

• Co-immunoprecipitation to identify stress-induced changes in PABP interaction partners

• Polysome profiling combined with western blotting to assess PABP association with translating ribosomes

• Crosslinking and immunoprecipitation (CLIP) to identify changes in PABP-bound mRNAs

• Subcellular fractionation followed by western blotting to quantify PABP distribution

These approaches collectively provide insights into how stress affects PABP function and localization, which directly impacts translational regulation mechanisms. Researchers should include appropriate controls for each stress condition and consider the timing of stress induction to capture both early and late events .

What approaches can be used to evaluate PAP-based vaccines for prostate cancer immunotherapy?

Evaluating PAP-based vaccine candidates requires a multi-faceted approach:

• In vitro stimulation of patient PBMCs with the candidate vaccine to assess immunogenicity

• Comparison of wild-type versus mutated PAP peptides (e.g., MutPAP42mer vs. wild-type)

• Testing different adjuvant combinations (e.g., CAF®09 vs. CpG ODN1826)

• Assessment of both CD4+ and CD8+ T cell responses to determine helper and cytotoxic effects

• Evaluation of memory T cell generation and persistence after stimulation

• Functional cytotoxicity assays against PAP-expressing tumor cells (e.g., LNCaP)

Research has demonstrated that mutated PAP sequences (e.g., changing alanine to leucine at position 116) can increase MHC binding scores and enhance immunogenicity. Additionally, longer peptides (e.g., 42mer vs. 15mer) can induce stronger CD8+ T cell reactivity through incorporation of multiple epitopes .

How can researchers investigate PABP involvement in viral infection mechanisms?

PABP plays a critical role during viral infections, particularly with viruses like poliovirus that cleave PABP to shut down host protein synthesis. Research methodologies to investigate this include:

• Time-course western blotting to monitor PABP cleavage patterns during infection

• Immunofluorescence to track changes in PABP localization during different infection stages

• Co-immunoprecipitation to identify viral proteins that interact with PABP

• Site-directed mutagenesis of PABP cleavage sites to create cleavage-resistant variants

• Polysome profiling to assess the impact of PABP cleavage on translation efficiency

• Rescue experiments expressing cleavage-resistant PABP to determine functional consequences

Understanding PABP dynamics during viral infection provides insights into viral strategies for host translation shutdown and may reveal therapeutic targets for antiviral development .

How should researchers troubleshoot non-specific binding or weak signals when using PABP Antibody (10E10)?

When facing non-specific binding or weak signal issues:

• For non-specific bands in western blotting:

  • Optimize antibody dilution (try 1:500 to 1:2000 range)

  • Increase blocking time and concentration (5-10% blocking agent)

  • Add 0.1-0.5% Tween-20 to wash buffers

  • Consider alternative blocking agents (milk vs. BSA)

  • Pre-absorb antibody with the blocking agent

• For weak signals:

  • Increase protein loading (50-100 μg)

  • Reduce washing stringency

  • Extend primary antibody incubation (overnight at 4°C)

  • Use signal enhancement systems (HRP-conjugated secondary antibodies)

  • Consider using more sensitive detection reagents

• For immunofluorescence:

  • Optimize fixation method (4% PFA vs. methanol)

  • Adjust permeabilization conditions (0.1-0.5% Triton X-100)

  • Test different antigen retrieval methods

  • Use directly conjugated antibody versions to reduce background

Include appropriate controls in all troubleshooting experiments to properly interpret results .

What are the key considerations when analyzing PAP-specific T cell responses in patient samples?

When analyzing PAP-specific T cell responses in patient samples, researchers should consider:

• Patient variation: HLA haplotype differences impact epitope recognition (use HLA typing)

• Prior treatments: Previous therapies may influence baseline immune responses

• Sample handling: Standardize PBMC isolation and cryopreservation protocols

• Background responses: Include unstimulated controls to determine baseline activation

• Positive controls: Use mitogen stimulation (PHA, PMA/ionomycin) to confirm cell viability

• Multi-parameter analysis: Assess not only frequency but also functionality of PAP-specific T cells

• Statistical approach: Account for donor variability and determine appropriate sample sizes

Flow cytometry using DextramerTM technology has been successfully employed to detect PAP-135-143 epitope-specific CD8+ T cells in prostate cancer patients. Additionally, functional assays demonstrating enhanced killing capacity against PAP-expressing LNCaP cells following stimulation with mutPAP42mer provide important correlates of potential clinical efficacy .

How can researchers interpret changes in PABP localization during stress and what controls are essential?

Interpreting PABP localization changes requires:

• Baseline characterization: Under normal conditions, PABP is predominantly cytoplasmic with diffuse distribution

• Stress-specific patterns: Different stressors cause distinct localization patterns

  • Arsenite: PABP relocalization to stress granules

  • Viral infection: Potential nuclear accumulation or degradation

  • Heat shock: Altered distribution between soluble and insoluble fractions

Essential controls include:

• Unstressed cells for baseline comparison

• Positive controls for each stress condition

• Co-staining with markers of specific cellular compartments (G3BP for stress granules, TIAR for P-bodies)

• Time-course analysis to distinguish transient from persistent changes

• Rescue experiments (e.g., overexpression of stress granule disassembly factors)

Quantitative analysis should include:

• Measurement of nuclear/cytoplasmic ratios

• Colocalization coefficients with organelle markers

• Granule size and number quantification

• Correlation with functional readouts (e.g., translation efficiency)

How might researchers utilize both PABP and PAP antibodies in combined diagnostic approaches?

Though PABP and PAP have distinct biological roles, researchers might explore combined approaches:

• Multiplex immunohistochemistry panels including both PABP and PAP antibodies to assess:

  • Translational activity (PABP) alongside tumor-specific markers (PAP) in prostate cancer

  • Correlation between translational dysregulation and PAP expression patterns

  • Prognostic value of combined biomarker assessment

• Development of liquid biopsy approaches detecting both:

  • Circulating tumor cells using PAP as a prostate-specific marker

  • Exosomal PABP patterns as indicators of translational reprogramming

• Therapeutic response monitoring:

  • PABP localization as an indicator of stress response to therapy

  • PAP-specific immune responses following immunotherapy

Such combined approaches require careful validation of antibody specificity and optimization of multiplexed detection protocols .

How could emerging PAP-based vaccine designs be improved based on recent immunological findings?

Building on current research findings, several strategies could improve PAP-based vaccine designs:

• Epitope optimization: Further refining amino acid substitutions beyond the alanine to leucine change at position 116 to enhance MHC binding and T cell receptor recognition

• Multi-epitope constructs: Designing chimeric peptides containing multiple PAP epitopes to broaden immune responses across diverse HLA types

• Novel adjuvant combinations: Testing CAF®09 in combination with other immune activators to enhance both CD4+ and CD8+ responses

• Delivery systems: Exploring nanoparticle formulations or viral vectors for improved antigen presentation

• Combination approaches: Integrating PAP vaccines with immune checkpoint inhibitors or other immunotherapies

• Personalized epitope selection: Tailoring epitope selection based on individual patient HLA haplotypes

Recent research demonstrating that mutated PAP sequences induce stronger immune responses than wild-type sequences provides a foundation for these optimization strategies .

What novel methods are emerging for studying PABP dynamics in living cells?

Several cutting-edge approaches are being developed to study PABP dynamics:

• Live-cell imaging techniques:

  • CRISPR-mediated endogenous tagging of PABP with fluorescent proteins

  • Photoactivatable or photoconvertible PABP fusions to track movement between compartments

  • FRAP (Fluorescence Recovery After Photobleaching) to measure PABP mobility

• Proximity labeling approaches:

  • BioID or TurboID fusions to map PABP interaction networks in different cellular states

  • APEX2-based labeling to identify transient PABP binding partners

• Single-molecule techniques:

  • smFISH combined with PABP immunofluorescence to visualize PABP-mRNA interactions

  • Super-resolution microscopy to resolve PABP distribution within subcellular compartments

  • Single-molecule tracking to determine PABP binding kinetics

• RNA-protein interaction mapping:

  • CLIP-seq variants to identify PABP binding sites genome-wide

  • RNA Bind-n-Seq to determine PABP binding preferences