PEBP1 Antibody

What are the primary applications for PEBP1 antibodies in research?

PEBP1 antibodies are primarily used for Western blot (WB) and immunofluorescence/immunocytochemistry (IF/ICC) applications in human samples . The antibody enables detection of PEBP1 protein (approximately 21-23 kDa) in various experimental contexts. Based on sequence alignment predictions, certain antibodies like the DF4290 rabbit polyclonal may also cross-react with PEBP1 from other species including pig, zebrafish, bovine, horse, sheep, rabbit, dog, chicken, and Xenopus . When designing experiments, researchers should consider:

• Primary application requirements (protein detection vs. localization)

• Sample type (cell lysates, tissue sections, cerebrospinal fluid)

• Detection method compatibility (colorimetric, chemiluminescent, fluorescent)

• Species compatibility based on the experimental model

In cerebrospinal fluid studies, sandwich ELISA has been successfully developed for PEBP1 detection, providing an additional quantitative application .

What precautions should be taken when handling samples for PEBP1 detection?

PEBP1 is highly sensitive to pre-analytical conditions, particularly prolonged storage at room temperature or 4°C . Researchers should implement the following sample handling protocols:

• Process samples immediately when possible

• Store samples at -80°C for long-term stability

• Avoid repeated freeze-thaw cycles

• Include protease and phosphatase inhibitors (particularly important due to PEBP1's multiple phosphorylation sites)

• Consider concentration techniques for low-abundance samples (e.g., Vivaspin® filtering for CSF samples)

For cerebrospinal fluid specifically, 5x concentration using appropriate molecular weight cut-off filters (e.g., 5 kDa) has been successfully employed to enhance detection sensitivity .

How should PEBP1 antibodies be validated before experimental use?

Before employing PEBP1 antibodies in critical experiments, validation through multiple approaches is recommended:

• Western blot confirmation with recombinant PEBP1 protein

• Testing across multiple matrices (neat and concentrated CSF pools, human brain homogenate, recombinant protein)

• Verification of specific binding in both reducing (WB) and native conditions (direct ELISA)

• Cross-reactivity assessment with abundant proteins (e.g., human serum albumin)

• Positive and negative control inclusion (PEBP1 knockout or knockdown samples)

When developing assays like ELISA, testing multiple antibody pairs (capture/detection combinations) at varied concentrations (e.g., 0.5-2μg/mL) optimizes signal-to-noise ratio .

What cell culture models are suitable for studying PEBP1 function?

Several cell lines have been validated for PEBP1 research, each with specific advantages depending on the research question:

All cell cultures should be maintained with appropriate antibiotics and regularly tested for mycoplasma contamination . For siRNA knockdown studies, transfection protocols using Lipofectamine 3000 in OptiMEM medium have demonstrated effective PEBP1 depletion .

How can researchers effectively measure PEBP1's role in the integrated stress response (ISR)?

To investigate PEBP1's role in mitochondrial-induced integrated stress response, a multi-parameter approach is recommended:

• eIF2α phosphorylation assessment: Western blotting for phospho-eIF2α (Ser51) as the primary ISR indicator

• ATF4 reporter activity: Utilizing ATF4 promoter-reporter constructs to measure downstream ISR activation

• Global protein synthesis measurement: L-homopropargylglycine (HPG) incorporation rather than puromycin assay (which is unreliable in energy-starved cells)

• Gene expression analysis: RT-qPCR for ISR-responsive genes

• Cellular response evaluation: Assessment of cell viability, morphology, and recovery after stress

When studying mitochondrial dysfunction specifically, oligomycin treatment provides a reliable model, with PEBP1 knockout showing partial rescue of protein synthesis rates under these conditions .

What methods are available for detecting PEBP1 protein-protein interactions?

PEBP1 functions through multiple protein interactions, particularly with the RAF/MEK/ERK pathway components. Several techniques have been validated:

• Real-time interaction analysis: Luminescence complementation in live cells has successfully revealed PEBP1-eIF2α interactions, including disruption upon eIF2α S51 phosphorylation

• Co-immunoprecipitation: Effective for detecting RAF1-PEBP1 interactions, enhanced when RAF1 is phosphorylated on Ser-338, Ser-339, Tyr-340, and Tyr-341

• Mass spectrometry-based cellular thermal shift assay (MS-CETSA): Revealed PEBP1 thermal stabilization by stresses inducing mitochondrial ISR

• Proximity ligation assays: For visualizing interactions in intact cells

• Yeast two-hybrid screening: For identifying novel interaction partners

These methods provide complementary information about PEBP1's dynamic interaction network and should be selected based on the specific research question.

How can post-translational modifications of PEBP1 be comprehensively analyzed?

PEBP1 undergoes extensive post-translational modifications that regulate its function. An integrated approach to PTM analysis includes:

A comprehensive PTM mapping would combine enrichment techniques (TiO₂ for phosphopeptides, antibody-based enrichment for acetylation/ubiquitination) with high-resolution mass spectrometry. Software tools like Proteome Discoverer or MaxQuant with PTM-focused search parameters enable comprehensive analysis .

What strategies can resolve conflicting data regarding PEBP1's role in disease biomarker applications?

PEBP1 has been investigated as a potential Alzheimer's disease biomarker, though with inconsistent results. To address conflicting findings:

• Standardize pre-analytical procedures: Given PEBP1's sensitivity to storage conditions, standardized sample handling protocols are essential

• Develop robust assay methods: Validated ELISA with carefully selected antibody pairs and reference standards

• Incorporate multi-marker panels: Analyze PEBP1 alongside established markers (Aβ₄₀, t-tau, p-tau)

• Patient stratification: Refine cohort selection criteria (disease stage, comorbidities)

• Statistical approaches: Use multivariate analysis to account for confounding variables

Research has shown weak positive correlations between CSF PEBP1 and t-tau, p-tau, and Aβ₄₀ in AD-dementia groups, suggesting PEBP1 may complement existing biomarkers rather than serve as a standalone indicator .

How can PEBP1's mitochondrial stress amplification role be therapeutically targeted?

PEBP1's recently identified role in amplifying mitochondrial stress signals suggests potential therapeutic applications. Research approaches include:

• Structure-based drug design: Target the PEBP1-eIF2α interaction interface

• Small molecule screening: Identify compounds that modulate PEBP1's effect on ISR activation

• Genetic approaches: CRISPR-based modulation of PEBP1 expression or specific functional domains

• Pathway-specific interventions: Target downstream effectors in mitochondrial ISR selectively

When investigating therapeutic potential, researchers should consider that complete PEBP1 inhibition may be detrimental, as PEBP1 facilitates appropriate cellular responses to acute mitochondrial dysfunction . Targeted modulation rather than complete inhibition may prove more effective for diseases with mitochondrial dysfunction components.

How can researchers optimize PEBP1 antibody selection for specific applications?

Antibody selection significantly impacts experimental outcomes. Consider these application-specific recommendations:

For sandwich ELISA development specifically, testing multiple antibody pairs at various concentrations (0.5-2μg/mL) helps identify optimal signal-to-noise ratios. Antibodies recognizing different regions of PEBP1 provide better assay performance .

What are effective strategies for studying PEBP1 in difficult-to-analyze samples like cerebrospinal fluid?

Cerebrospinal fluid presents unique challenges for PEBP1 analysis:

• Sample concentration: Use 5kDa molecular weight cut-off filters for 5x concentration of CSF samples

• Protein quantification: Bradford assay provides reliable total protein estimation in neat and concentrated CSF

• Storage optimization: Immediate processing and storage at -80°C

• Internal standards: Include recombinant PEBP1 standards calibrated against reference materials

• Matrix effects mitigation: Sample dilution series to identify potential interfering factors

In clinical validation studies, researchers should account for patient heterogeneity by carefully defining inclusion/exclusion criteria and collecting detailed clinical data .

How should researchers design experiments to distinguish PEBP1's ISR-specific functions from its RAF/MEK/ERK pathway effects?

PEBP1 has dual roles in ISR activation and RAF/MEK/ERK pathway inhibition. To distinguish these functions:

• Domain-specific mutants: Generate PEBP1 variants with selective disruption of specific interaction surfaces

• Pathway-specific readouts: Monitor both phospho-eIF2α (ISR) and phospho-ERK (MAPK pathway) simultaneously

• Selective pathway activators/inhibitors: Use RAF/MEK/ERK inhibitors to isolate ISR-specific effects

• Time-course experiments: Different temporal dynamics may distinguish the pathways

• Cell-type specific analyses: Some cell types may predominately utilize one PEBP1 function

Research has demonstrated that PEBP1's role in ISR activation during mitochondrial stress is independent of its function in inhibiting the RAF/MEK/ERK pathway , highlighting the importance of pathway-specific controls in experimental design.

How can advanced imaging techniques enhance understanding of PEBP1's subcellular dynamics?

Emerging imaging approaches offer new insights into PEBP1 biology:

• Super-resolution microscopy: Techniques like STORM or PALM provide nanoscale resolution of PEBP1 localization

• Live-cell imaging: PEBP1-fluorescent protein fusions for real-time tracking during stress responses

• FRET/BRET approaches: Measure dynamic PEBP1 interactions with binding partners

• Correlative light-electron microscopy: Connect PEBP1 localization with ultrastructural features

• Spatial transcriptomics: Correlate PEBP1 protein localization with local transcriptional responses

These approaches can reveal how PEBP1 translocation and interaction dynamics change during cellular stress responses, particularly in relation to mitochondrial function and integrated stress response activation .

What bioinformatic approaches can reveal insights from PEBP1 multi-omics data integration?

To fully understand PEBP1's role in cellular homeostasis and disease, multi-omics integration approaches include:

• Network analysis: Construct protein-protein interaction networks centered on PEBP1

• Pathway enrichment: Identify overrepresented pathways in PEBP1-associated genes/proteins

• Multi-omics correlation: Connect PEBP1 expression/modification with transcriptomic, proteomic, and metabolomic changes

• Machine learning approaches: Predict PEBP1 functions based on integrated datasets

• Evolutionary analysis: Compare PEBP1 conservation and divergence across species

These computational approaches can guide experimental design by generating testable hypotheses about PEBP1's role in complex biological processes and disease mechanisms.