PEA15 Antibody

What is PEA-15 and why is it significant in cellular research?

PEA-15 (Phosphoprotein Enriched in Astrocytes, 15 kDa) is a small, death effector domain (DED)-containing protein that plays critical roles in regulating mitogen-activated protein kinase signaling and apoptosis pathways. Research significance stems from its multifunctional nature - PEA-15 inhibits tumor necrosis factor-α-induced apoptosis and reverses H-Ras-mediated inhibition of integrin activation . PEA-15's involvement in cellular signaling makes it a valuable target for understanding disease mechanisms, particularly in cancer research where it has been shown to confer resistance to sorafenib therapy and promote migration in hepatocellular carcinoma . The protein's ability to switch binding partners based on phosphorylation state (from ERK to FADD) represents a paradigmatic example of how post-translational modifications can drastically alter protein function and cellular outcomes .

What are the primary applications for PEA-15 antibodies in molecular biology research?

PEA-15 antibodies serve multiple critical applications in molecular biology research. Western blotting applications have been validated with specific detection of PEA-15 at approximately 15 kDa in cellular lysates (e.g., A172 human glioblastoma cell line and human cortex tissue) . Immunohistochemistry applications show reliable detection in paraffin-embedded tissue sections, with validated protocols demonstrating specific staining localized to neurons in human brain cortex samples . Cell-based assays examining protein expression and functional roles can benefit from PEA-15 antibodies in immunofluorescence and immunoprecipitation applications. The availability of both polyclonal and monoclonal antibodies allows researchers to select the appropriate reagent based on their specific experimental needs . When designing experiments, researchers should consider antibody specificity for human/mouse PEA-15 (such as the AF5588 antibody that recognizes recombinant human PEA-15 Ala2-Ala130) .

How should researchers select the appropriate PEA-15 antibody for their specific experimental application?

Selection of an appropriate PEA-15 antibody should be based on several key experimental factors. Consider the host species compatibility - using antibodies raised in species distinct from your experimental model (e.g., sheep anti-human/mouse or mouse monoclonal antibodies) to avoid cross-reactivity . Evaluate epitope recognition requirements - for phosphorylation studies, select antibodies that either recognize or are unaffected by the phosphorylation status at Ser-104 and Ser-116, critical regulatory sites that alter PEA-15 function . The experimental application dictates specific antibody requirements - for Western blotting, validated antibodies like the sheep anti-human/mouse have demonstrated detection of the ~15 kDa PEA-15 band under reducing conditions . For immunohistochemistry on paraffin-embedded sections, antibodies validated for antigen retrieval compatibility are essential, such as those demonstrated to work with overnight incubation at 4°C . Importantly, when studying PEA-15 interactions with binding partners like ERK or FADD, ensure the selected antibody's epitope doesn't interfere with these protein-protein interaction sites .

What are the optimal conditions for using PEA-15 antibodies in Western blot applications?

Optimal Western blotting with PEA-15 antibodies requires careful protocol optimization. Based on validated protocols, researchers should prepare samples under reducing conditions, which has been shown to effectively detect the 15 kDa PEA-15 protein . For membrane preparation, PVDF membranes have demonstrated good results in published protocols. Primary antibody concentration optimization is critical - for the sheep anti-human/mouse PEA-15 antibody (AF5588), a concentration of 1 μg/mL has been validated . Secondary antibody selection should match the host species of your primary antibody - for example, HRP-conjugated anti-sheep IgG works well with sheep-derived primary antibodies . Buffer selection impacts detection sensitivity - Immunoblot Buffer Group 8 has been specifically validated for PEA-15 detection . When analyzing results, expect visualization of a specific band at approximately 15 kDa, which corresponds to the molecular weight of PEA-15 . For challenging samples or weak signals, researchers may need to increase protein loading or extend antibody incubation times.

How can researchers effectively use PEA-15 antibodies in immunohistochemistry and immunofluorescence studies?

Effective immunohistochemistry with PEA-15 antibodies requires multiple optimization steps. For fixation, immersion-fixed paraffin-embedded sections have demonstrated successful staining, particularly in human brain cortex samples . Antigen retrieval is crucial for paraffin sections - though specific protocols weren't detailed in the search results, heat-induced epitope retrieval is typically necessary for intracellular proteins like PEA-15. Primary antibody concentration has been validated at 1 μg/mL for overnight incubation at 4°C with the sheep anti-human/mouse PEA-15 antibody . Detection systems such as the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit have produced specific brown staining of neurons in brain tissue . Counterstaining with hematoxylin provides blue nuclear staining that contrasts well with the brown DAB signal . For expected staining patterns, PEA-15 demonstrates specific localization to neurons in brain tissue, which serves as a positive control for staining specificity . For immunofluorescence applications, though not explicitly detailed in the search results, researchers should select fluorophore-conjugated secondary antibodies compatible with their microscopy setup and consider dual-staining approaches to examine co-localization with binding partners like ERK or FADD.

What are the recommended protocols for immunoprecipitation using PEA-15 antibodies?

While the search results don't provide explicit immunoprecipitation protocols for PEA-15, successful immunoprecipitation approaches can be derived from general principles and mentioned applications. For antibody selection, both monoclonal and polyclonal antibodies have been generated against PEA-15, with monoclonal antibodies often providing higher specificity while polyclonals may capture more protein variants . Researchers should consider testing multiple antibodies as immunoprecipitation efficiency varies between clones. For bead selection, protein A/G beads are commonly used for rabbit polyclonal antibodies, while specific anti-mouse or anti-sheep IgG beads may be optimal for monoclonal or sheep-derived antibodies respectively . Cell lysis conditions should preserve protein-protein interactions - for studies examining PEA-15 interactions with ERK or FADD, non-denaturing lysis buffers are recommended . Elution strategies should be optimized based on downstream applications - acidic glycine elution preserves antibody on beads, while boiling in SDS sample buffer elutes both target and antibody. For validation, Western blot analysis using a separate PEA-15 antibody raised in a different species can confirm successful immunoprecipitation . Co-immunoprecipitation approaches can specifically investigate PEA-15 interactions with binding partners like ERK and FADD, which switch based on phosphorylation status .

How can researchers effectively use PEA-15 antibodies to study its phosphorylation-dependent interactions?

PEA-15 phosphorylation status critically determines its binding partners and cellular functions, making phospho-specific approaches essential. Phosphorylation site mapping reveals that PEA-15 has two key phosphorylation sites at Ser-104 and Ser-116, which allosterically mediate conformational changes of the DED domain . Binding partner switching occurs where unphosphorylated PEA-15 preferentially binds ERK, while doubly-phosphorylated PEA-15 binds to FADD . This represents a fundamental regulatory mechanism for PEA-15 function.

To effectively study these phosphorylation-dependent interactions, researchers should implement a multi-faceted approach:

• Utilize phosphorylation-state specific antibodies (if available) that specifically recognize unphosphorylated, singly-phosphorylated (either Ser-104 or Ser-116), or doubly-phosphorylated PEA-15

• Employ site-directed mutagenesis to create phosphomimetic (S104D/S116D) or phospho-deficient (S104A/S116A) PEA-15 mutants for functional studies

• Combine co-immunoprecipitation with phospho-specific antibodies to isolate and identify binding partners under different phosphorylation conditions

• Design pull-down assays using recombinant PEA-15 with controlled phosphorylation states to examine binding affinities with purified ERK2 and FADD

• Implement microscopy approaches with phospho-specific antibodies to visualize subcellular localization changes dependent on phosphorylation state

Through molecular dynamics studies, researchers have revealed that "the binding interfaces between the unphosphorylated PEA-15 and ERK2 and between the doubly phosphorylated PEA-15 and FADD are similarly composed of a scaffold that includes both the DED and the C-terminal tail residues of PEA-15" . This provides a structural basis for designing experiments to probe these interactions.

What approaches can researchers use to study PEA-15's role in cancer biology, particularly regarding therapeutic resistance?

PEA-15 has significant implications in cancer biology, particularly regarding therapeutic resistance to sorafenib in hepatocellular carcinoma (HCC). To investigate these functions, researchers should employ a comprehensive experimental approach:

For expression analysis, quantify PEA-15 levels across cancer cell lines using validated antibodies - as demonstrated in the study showing variable PEA-15 expression across 10 different HCC cell lines . Functional manipulation experiments should include both overexpression and knockdown approaches - transfection with PED-MYC vector for overexpression and siRNA against PED for silencing have been successfully implemented .

When assessing therapeutic resistance, researchers should measure cell viability after drug treatment in cells with modulated PEA-15 expression - MTT assays at 24h and 48h timepoints after sorafenib treatment have revealed PEA-15-dependent resistance patterns . Apoptosis assays should quantify caspase-3/7 activation following drug treatment - studies have shown that PEA-15 silencing enhances sorafenib-induced caspase-3 activation . For real-time monitoring, systems like xCELLigence provide continuous assessment of cell growth properties in response to treatment and PEA-15 modulation .

This table summarizes key experimental conditions and findings regarding PEA-15's role in sorafenib resistance .

How can researchers integrate PEA-15 antibody-based approaches with molecular dynamics studies to understand structure-function relationships?

Integrating antibody-based experimental approaches with molecular dynamics (MD) simulations provides powerful insights into PEA-15's structure-function relationships. MD studies have revealed that "phosphorylation of the PEA-15 C-terminal tail residues, Ser-104 and Ser-116, allosterically mediates conformational changes of the DED and alters the binding specificity from extracellular-regulated kinase (ERK) to Fas-associated death domain (FADD) protein" .

To leverage these insights experimentally, researchers should:

• Design epitope mapping experiments using a panel of antibodies that recognize distinct regions of PEA-15 to experimentally validate conformational changes predicted by MD simulations

• Employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) with antibody-based enrichment to identify regions with altered solvent accessibility upon phosphorylation

• Utilize conformation-specific antibodies (if available) that selectively recognize the ERK-binding or FADD-binding conformations of PEA-15

• Perform cross-linking mass spectrometry with antibody-based pulldowns to capture interaction interfaces between PEA-15 and its binding partners

• Design antibody-based FRET sensors to monitor conformational changes in live cells upon stimulation of pathways that regulate PEA-15 phosphorylation

MD simulations have revealed specific interaction details, such as "the phosphorylated Ser-116 engages in strong electrostatic interactions with arginine residues on FADD DED" . Researchers can test these predictions using site-directed mutagenesis of these arginine residues followed by antibody-based detection of binding.

Additionally, the finding that "upon PEA-15 binding, FADD repositions its death domain (DD) relative to the DED, an essential conformational change to allow the death-inducing signaling complex (DISC) assembly" suggests that researchers should examine downstream DISC formation using antibody-based approaches following manipulation of PEA-15 phosphorylation state.

What are common challenges when using PEA-15 antibodies and how can researchers address them?

Researchers working with PEA-15 antibodies may encounter several challenges that require systematic troubleshooting approaches. For non-specific binding issues, researchers should implement more stringent blocking conditions, such as 5% BSA or milk in TBS-T, and consider titrating primary antibody concentrations - the validated concentration of 1 μg/mL for the sheep anti-human/mouse antibody provides a starting point . When facing weak signal problems, researchers may need to optimize protein extraction protocols to preserve PEA-15, as phosphatases can rapidly dephosphorylate key regulatory sites. For inconsistent results between experiments, standardize incubation times and temperatures - overnight incubation at 4°C has been validated for immunohistochemistry applications .

Cross-reactivity concerns with other DED-containing proteins can occur since PEA-15 contains a death effector domain shared by other proteins. To address this, researchers should validate antibody specificity through knockout/knockdown controls. For detecting phosphorylated forms, phosphatase inhibitors must be included in all steps of sample preparation, and phospho-specific antibodies should be validated with phosphatase-treated negative controls. When interpreting subcellular localization data, researchers should be aware that PEA-15 localization may vary by cell type - neuronal localization has been validated in brain tissue sections , but other cell types may show different patterns.

How can researchers validate the specificity and functionality of PEA-15 antibodies?

Rigorous validation of PEA-15 antibodies ensures experimental reliability. Knockout/knockdown controls provide the gold standard - siRNA against PEA-15/PED has been successfully used in multiple cell lines including HuH-7 and SNU-449, making these useful systems for antibody validation . Western blot analysis should confirm detection of a single band at approximately 15 kDa in positive control samples like A172 human glioblastoma cell line or human cortex tissue . Multiple antibody comparison can enhance confidence - using both monoclonal and polyclonal antibodies against different epitopes should yield consistent results .

Phosphorylation-state validation is critical when studying PEA-15 biology - treatment with phosphatases should eliminate signal from phospho-specific antibodies. Immunoprecipitation-mass spectrometry provides definitive validation by confirming the identity of the immunoprecipitated protein. Cross-reactivity assessment with related proteins, particularly other DED-containing proteins, should be conducted to ensure specificity. For immunohistochemistry applications, researchers should compare staining patterns with published results - specific neuronal staining in brain tissue has been documented and can serve as a positive control . Pre-absorption controls with recombinant PEA-15 protein can confirm specificity by blocking antibody binding to the target in tissue sections.

What considerations should researchers take when analyzing conflicting data from PEA-15 antibody-based experiments?

When faced with conflicting experimental results using PEA-15 antibodies, systematic analysis is essential. Phosphorylation state variations are a primary concern since PEA-15 function dramatically changes based on phosphorylation at Ser-104 and Ser-116 . Researchers should carefully document and control phosphorylation status in their experimental systems, as differences may explain conflicting results. Cell type-specific effects should be considered - PEA-15 expression varies significantly across different cell lines, as demonstrated in the analysis of 10 different HCC cell lines . This variation may contribute to different functional outcomes.

Protocol differences between labs can significantly impact results - even minor variations in antibody concentration, incubation time, or buffer composition can affect detection sensitivity and specificity. For example, validated protocols for the sheep anti-human/mouse antibody specify 1 μg/mL concentration and particular buffer conditions . Isoform detection variability may occur if different antibodies recognize distinct epitopes that may be affected by splice variants or post-translational modifications. When integrating data from multiple approaches, researchers should ensure complementary methods validate key findings - combining Western blot, immunoprecipitation, and functional assays provides stronger evidence than any single approach.

Data interpretation requires careful consideration of controls - studies manipulating PEA-15 expression demonstrate the importance of both overexpression and knockdown controls to establish causality . When publishing, researchers should clearly document antibody validation methods, experimental conditions, and any observed variability to help the field reconcile apparently conflicting results.

How might emerging technologies enhance PEA-15 antibody-based research?

Emerging technologies offer promising avenues to advance PEA-15 antibody-based research. Single-cell protein analysis techniques, including mass cytometry (CyTOF) and single-cell Western blotting, could reveal heterogeneity in PEA-15 expression and phosphorylation states within populations, potentially uncovering subpopulations with distinct signaling profiles. Proximity labeling approaches like BioID or APEX when combined with PEA-15 antibodies could map the dynamic interactome of PEA-15 under different cellular conditions and phosphorylation states, expanding our understanding beyond the known ERK and FADD interactions .

Super-resolution microscopy techniques (STORM, PALM, SIM) with fluorophore-conjugated PEA-15 antibodies could visualize nanoscale spatial organization of PEA-15 with unprecedented detail, potentially revealing functional microdomains. Phospho-specific biosensors based on FRET or bioluminescence may allow real-time monitoring of PEA-15 phosphorylation states in living cells, providing temporal information about signaling dynamics. CRISPR-Cas9 genome editing to introduce epitope tags at the endogenous PEA-15 locus would enable antibody-based detection of PEA-15 at physiological expression levels, avoiding artifacts associated with overexpression systems described in current research .

Microfluidic antibody-based assays could provide high-throughput screening of PEA-15 expression, phosphorylation, and protein interactions across numerous conditions simultaneously. Advanced computational approaches integrating antibody-based proteomics data with molecular dynamics simulations would create more comprehensive models of PEA-15 structure-function relationships, building upon current findings about phosphorylation-dependent conformational changes .

What are the most promising therapeutic applications of research utilizing PEA-15 antibodies?

Research utilizing PEA-15 antibodies reveals several promising therapeutic directions. In cancer therapy resistance, PEA-15 has been identified as conferring resistance to sorafenib in hepatocellular carcinoma . Therapeutic approaches targeting PEA-15 could potentially overcome this resistance mechanism - antibody-based screening assays could identify small molecules that disrupt PEA-15's pro-survival functions or enhance its degradation. For metastasis prevention, research shows PEA-15 "promotes migration in hepatocellular carcinoma" , suggesting that inhibiting PEA-15 might reduce metastatic potential - antibody-based approaches could help validate this as a therapeutic strategy and identify intervention points.

In apoptosis modulation, PEA-15 "inhibits tumor necrosis factor-α-induced apoptosis" , indicating that targeting PEA-15 might sensitize resistant cancer cells to apoptotic stimuli. Antibody-based assays could screen for compounds that interfere with this anti-apoptotic function. The phosphorylation-dependent switching between ERK and FADD binding partners suggests that selectively modulating PEA-15 phosphorylation could redirect cellular signaling either toward proliferation or apoptosis, depending on the therapeutic goal. Developing kinase inhibitors that specifically affect PEA-15 phosphorylation could be screened using phospho-specific antibodies.

As a diagnostic biomarker, antibody-based detection of PEA-15 expression or phosphorylation status might predict treatment response or prognosis, particularly in cancers where PEA-15 mediates therapeutic resistance . For combination therapy approaches, understanding how PEA-15 interacts with other cellular pathways could inform rational drug combinations - antibody-based proteomics could map these interactions and identify synergistic targets.

How can researchers contribute to standardization of PEA-15 antibody-based methods in the scientific community?

Standardization of PEA-15 antibody-based methods would accelerate research progress and enhance reproducibility across laboratories. To contribute to this effort, researchers should implement detailed antibody validation reporting in publications, including complete information about antibody source, catalog number, lot number, validation methods used, and observed limitations. For example, when using the sheep anti-human/mouse PEA-15 antibody (Catalog # AF5588), researchers should document all validation steps performed .

Developing standard operating procedures (SOPs) for common applications would enhance consistency - detailed protocols for Western blotting, immunohistochemistry, and immunoprecipitation with PEA-15 antibodies should specify critical parameters like the validated 1 μg/mL antibody concentration for Western blot and IHC applications . Creating and sharing reference materials, such as recombinant PEA-15 proteins with defined phosphorylation states and PEA-15 knockout cell lines, would provide universal positive and negative controls for antibody validation.

Establishing antibody performance metrics through quantitative benchmarking of sensitivity, specificity, and reproducibility across different PEA-15 antibodies would help researchers select optimal reagents for specific applications. Implementing digital repositories for antibody validation data would allow researchers to share validation results and experimental conditions that affect antibody performance. Multi-laboratory ring trials comparing PEA-15 antibody performance across different labs using standardized samples and protocols could identify sources of variability and establish best practices.

Integrating antibody metadata into data repositories when publishing PEA-15 research would create a more comprehensive knowledge base. For example, researchers could document that monoclonal antibodies generated using DNA immunization required only 5 μg of DNA and resulted in class-switched, affinity-matured antibodies in less than 4 weeks . This approach to standardization would enhance research quality and accelerate scientific progress in understanding PEA-15 biology and its therapeutic implications.