Phospho-EIF4E (S209) Recombinant Monoclonal Antibody

What is Phospho-EIF4E (S209) and why is it important in cellular signaling?

Phospho-EIF4E (S209) refers to the eukaryotic translation initiation factor 4E (eIF4E) protein when phosphorylated at serine residue 209. eIF4E is a critical component of the translation initiation complex that binds to the 5' cap structure of mRNAs, facilitating their translation into proteins. The phosphorylation at S209 is a key regulatory event in the mTOR signaling pathway . This post-translational modification enhances eIF4E's affinity for the cap structure and promotes translation of specific mRNAs involved in cell proliferation, survival, and oncogenesis.

eIF4E is also known by several alternative names including EIF4EL1, EIF4F, eIF-4F 25 kDa subunit, and mRNA cap-binding protein . The importance of this phosphorylation lies in its role as a convergence point for multiple signaling pathways that regulate protein synthesis in response to various stimuli. Dysregulation of eIF4E phosphorylation has been implicated in numerous pathological conditions, particularly cancer, making it both a valuable research target and potential therapeutic biomarker.

How does a Phospho-EIF4E (S209) Recombinant Monoclonal Antibody work?

Phospho-EIF4E (S209) recombinant monoclonal antibodies, such as EP2151Y , are engineered to specifically recognize and bind to eIF4E only when it is phosphorylated at serine 209. These antibodies are developed using recombinant technology, which ensures batch-to-batch consistency and high specificity compared to traditional monoclonal or polyclonal antibodies.

The antibody contains a variable region that precisely recognizes the three-dimensional epitope created by the phosphorylated S209 residue and its surrounding amino acid sequence. The specificity is typically achieved through immunization with a synthetic phosphopeptide corresponding to the region surrounding Ser209 of human eIF4E. This design enables the antibody to discriminate between phosphorylated and non-phosphorylated forms of eIF4E with high selectivity.

In experimental applications, the antibody binds to phosphorylated eIF4E in samples, and this binding is then detected through various methods depending on the application (e.g., secondary antibodies in Western blots or immunofluorescence, direct detection in TR-FRET assays). The signal intensity correlates with the amount of phosphorylated eIF4E present, allowing both qualitative and quantitative assessments.

What are the primary applications for Phospho-EIF4E (S209) antibodies in research?

Phospho-EIF4E (S209) antibodies are versatile tools employed across multiple research applications:

Western Blot (WB): This is one of the most common applications, allowing quantitative analysis of eIF4E phosphorylation levels in cell or tissue lysates. The recombinant monoclonal antibody EP2151Y has been extensively validated for this application across multiple species .

Immunocytochemistry/Immunofluorescence (ICC/IF): These techniques enable visualization of the subcellular localization of phosphorylated eIF4E, providing insights into spatial regulation. Both EP2151Y and polyclonal antibodies have demonstrated utility in this application .

Immunoprecipitation (IP): This application allows isolation of phosphorylated eIF4E and its associated proteins, facilitating studies of protein-protein interactions influenced by this phosphorylation .

Dot Blot: A rapid screening method that can be used for quick assessment of phosphorylation status without the need for electrophoretic separation .

TR-FRET Assays: Homogeneous, high-throughput detection methods like THUNDER™ and HTRF allow quantitative measurement of eIF4E phosphorylation in cell-based experiments without washing steps, making them ideal for drug screening applications .

Each of these applications provides different insights into the regulation, function, and dynamics of eIF4E phosphorylation, allowing researchers to address diverse experimental questions from mechanistic studies to high-throughput screens.

What are the optimal conditions for Western Blot using Phospho-EIF4E (S209) antibodies?

Optimal Western blotting conditions for Phospho-EIF4E (S209) antibodies require careful attention to several parameters:

Sample Preparation:

• 10-20 μg of total protein per lane is typically sufficient

• Critical inclusion of phosphatase inhibitors (sodium fluoride, sodium orthovanadate) in lysis buffers to preserve phosphorylation

• Rapid processing of samples at cold temperatures to minimize phosphorylation loss

Blocking Conditions:

• 5% non-fat dry milk (NFDM) in TBST has been validated as effective

• Maintain consistent blocking conditions during both blocking and antibody incubation steps

Antibody Dilutions:

• Primary antibody (EP2151Y/ab76256): Ranges from 1/1,000 to 1/100,000 depending on the sample and detection method

• For standard applications, 1/1,000 dilution in 5% NFDM/TBST works well for most samples

• For strong signals (e.g., stimulated samples), higher dilutions (1/50,000 or 1/100,000) may be necessary to prevent oversaturation

• Secondary antibody (e.g., Goat Anti-Rabbit IgG H&L (HRP)): Typically used at 1/10,000 to 1/20,000 dilution

Detection and Exposure:

• Predicted band size for eIF4E is approximately 25 kDa

• Exposure times vary from 15 seconds to 3 minutes depending on signal strength

• Short exposures (15-30 seconds) are often sufficient for stimulated samples

• Longer exposures (1-3 minutes) may be required for basal phosphorylation detection

Controls:

• Always include appropriate positive controls (e.g., EGF or serum-stimulated cell lysates)

• Include negative controls such as phosphatase-treated lysates to confirm signal specificity

• Consider running total eIF4E detection in parallel for normalization purposes

How should samples be prepared for Immunocytochemistry/Immunofluorescence with Phospho-EIF4E (S209) antibodies?

For optimal Immunocytochemistry/Immunofluorescence (ICC/IF) with Phospho-EIF4E (S209) antibodies, follow these sample preparation guidelines:

Cell Preparation and Fixation:

• 100% methanol fixation has been validated for preserving phospho-epitopes of eIF4E

• Cell density should be optimized to allow clear visualization of individual cells

• For adherent cells like HEK293, coating plates with poly-L-lysine may improve attachment

Antibody Dilutions and Incubations:

• Primary phospho-eIF4E (S209) antibody: Typically used at 1/500 dilution

• Secondary antibody (e.g., Alexa Fluor® 488-conjugated anti-rabbit IgG): Used at 1/1,000 dilution

• Incubate antibodies in appropriate blocking buffer to minimize background

• Optimal incubation times are typically 1-2 hours at room temperature or overnight at 4°C for primary antibodies

Counterstaining and Controls:

• DAPI is recommended for nuclear visualization

• Additional markers such as alpha-tubulin can provide cytoskeletal reference

• Include a pan-eIF4E antibody (e.g., Y448/ab33766) in parallel samples to assess total protein localization versus phosphorylated form

Treatment Controls for Validation:

• Serum starvation followed by serum stimulation (20%) can be used to modulate eIF4E phosphorylation for positive controls

• Phosphatase inhibitor treatment can enhance phospho-specific signals

• The search results demonstrate that 20% serum treatment increases cytoplasmic staining of phospho-eIF4E in NIH/3T3 cells

Imaging Considerations:

How does the TR-FRET assay for Phospho-EIF4E (S209) detection work?

The TR-FRET (Time-Resolved Förster Resonance Energy Transfer) assay for Phospho-EIF4E (S209) detection is a homogeneous, no-wash immunoassay designed for high-throughput screening and quantitative analysis. This sophisticated technique operates through the following mechanism:

Assay Principle:

• The assay employs two specialized antibodies: one labeled with a donor fluorophore (Europium chelate; Eu-Ab1) that recognizes phosphorylated S209 on eIF4E, and another labeled with a far-red acceptor fluorophore (FR-Ab2) that binds to an invariant epitope of eIF4E

• When both antibodies bind to the same eIF4E molecule, their proximity enables energy transfer from the donor to the acceptor molecule

• Excitation of the Europium chelate (320-340 nm) triggers FRET to the acceptor, which emits at 665 nm

• Residual energy from the Eu chelate generates light at 615 nm

• The ratio of 665 nm/615 nm signal is used for quantification, which normalizes for well-to-well variations

Workflow (3-step process):

• Cell treatment: Cells are cultured in a microplate and treated with compounds of interest

• Cell lysis: Media is removed and cells are lysed with a specialized lysis buffer containing phosphatase inhibitors (sodium fluoride at 1 mM and sodium orthovanadate at 2 mM), followed by a 30-minute incubation

• Protein detection: Cell lysate (15 μL) is transferred to a detection plate, the antibody mix (5 μL) is added, followed by a 4-hour incubation before TR-FRET signal reading

Technical Advantages:

• Homogeneous format eliminates wash steps, reducing variability and simplifying automation

• Time-resolved detection minimizes background fluorescence by measuring long-lived emission signals

• Ratiometric readout (665/615 nm) provides internal normalization

• High sensitivity due to signal amplification through the FRET process

• The validated protocol demonstrates robust performance with Z'-factor values >0.6, indicating excellent assay quality for screening applications

What cell treatments effectively modulate eIF4E S209 phosphorylation?

Several cell treatments can effectively modulate eIF4E S209 phosphorylation, allowing researchers to establish experimental models with controlled phosphorylation states:

Treatments that Increase Phosphorylation:

Treatments that Decrease Phosphorylation:

Protocol Considerations:

• Optimization of dose and time is essential for each cell type

• For HEK293 cells, EGF at 3 nM for 30 minutes provides robust phosphorylation, sufficient for assay development and inhibitor screening

• Pre-treatment with inhibitors (e.g., CGP 57380) for 30 minutes before stimulation allows assessment of inhibitory effects

• The baseline phosphorylation state (affected by serum starvation protocols) influences the magnitude of response

• Phosphatase inhibitors should be included in lysis buffers (typically sodium fluoride at 1 mM and sodium orthovanadate at 2 mM)

These validated treatments provide researchers with reliable tools for establishing positive and negative controls, developing assays, and investigating the regulation of eIF4E phosphorylation across different experimental contexts.

How can I validate the specificity of Phospho-EIF4E (S209) antibody signals?

Validating the specificity of Phospho-EIF4E (S209) antibody signals is crucial for ensuring reliable and reproducible results. Multiple complementary approaches can be employed:

Phosphatase Treatment Control:

• Treat a portion of your lysate with alkaline phosphatase to remove phosphorylation

• The search results demonstrate that phosphatase-treated 293 cell lysates show significant reduction in signal with phospho-eIF4E (S209) antibody, confirming phospho-specificity

• This serves as a definitive negative control for phospho-specific detection

Stimulation/Inhibition Controls:

• Compare unstimulated cells with those treated with known inducers of eIF4E phosphorylation (e.g., EGF, serum, dexamethasone)

• Include samples treated with specific inhibitors like CGP 57380 that block the responsible kinases (MNK1/2)

• The dose-dependent response to inhibitors provides further validation of signal specificity

Dual Detection Approach:

• Perform parallel detection with a phospho-specific and a pan-eIF4E antibody

• The search results recommend using Anti-eIF4E antibody [Y448] (ab33766) as a pan control for the phospho-specific antibody

• Changes in phosphorylation should be detectable with the phospho-specific antibody while total protein levels (detected by the pan antibody) remain constant

• This approach confirms that signal changes reflect phosphorylation state rather than protein expression levels

Methodological Cross-Validation:

• Confirm findings using multiple detection techniques

• For example, validate Western blot results with immunofluorescence or TR-FRET assays

• The search results show consistency of results across Western blotting and immunofluorescence methods

Quantitative Validation:

• For TR-FRET assays, Z'-factor determination between untreated and treated conditions provides statistical validation of assay performance

• The search results report a Z'-factor of 0.68 for EGF-stimulated versus inhibitor-treated HEK293 cells, indicating excellent assay specificity and reproducibility

Why might I observe weak or no signal when using Phospho-EIF4E (S209) antibodies?

Several factors can contribute to weak or absent signals when using Phospho-EIF4E (S209) antibodies. Understanding and addressing these issues is essential for successful experiments:

Phosphorylation State Issues:

• Insufficient baseline phosphorylation: Without stimulation, some cell types may have very low levels of eIF4E S209 phosphorylation

• Loss of phosphorylation during sample preparation: Inadequate phosphatase inhibitors in lysis buffers can result in rapid dephosphorylation

• The search results emphasize the importance of phosphatase inhibitors like sodium fluoride (1 mM) and sodium orthovanadate (2 mM) in lysis buffers

Antibody-Related Factors:

• Suboptimal antibody dilution: The search results show that antibody concentrations vary widely depending on the application (1/1,000 to 1/100,000 for Western blotting)

• Inadequate incubation time: The TR-FRET assay specifies a 4-hour incubation for optimal detection

• Antibody degradation due to improper storage or repeated freeze-thaw cycles

Protocol-Specific Issues:

For Western Blotting:

• Inefficient protein transfer, particularly for smaller proteins like eIF4E (25 kDa)

• Improper blocking: The search results recommend 5% non-fat dry milk in TBST for blocking and antibody dilution

• Suboptimal detection settings: Exposure times for Western blots range from 15 seconds to 3 minutes depending on signal strength

For Immunofluorescence:

• Fixation method incompatible with phospho-epitope preservation

• Excessive permeabilization leading to epitope loss

• Inadequate antibody penetration into fixed cells

For TR-FRET Assays:

• Suboptimal cell density: The protocols specify 50,000 cells/well for HEK293 cells

• Insufficient lysis: Incomplete cell disruption reduces available epitopes

• Plate reader settings not optimized for TR-FRET detection

Experimental Timing:

• Transient phosphorylation: eIF4E S209 phosphorylation may peak and decline rapidly; optimization of timepoints is essential

• The search results indicate 30 minutes as an effective time point for EGF stimulation in HEK293 cells

What should I do if I observe unexpected band sizes in Western blots with Phospho-EIF4E (S209) antibodies?

Encountering unexpected band sizes in Western blots with Phospho-EIF4E (S209) antibodies requires systematic troubleshooting:

Expected Band Size Reference:

• The predicted molecular weight of eIF4E is approximately 25 kDa

• This should be the primary band observed in validated positive controls

For Higher Molecular Weight Bands:

• Post-translational modifications: Additional modifications beyond phosphorylation (e.g., ubiquitination, SUMOylation) can increase apparent molecular weight

• Protein complexes: Incomplete denaturation may preserve eIF4E interactions with binding partners

• Solution: Ensure thorough sample denaturation with adequate SDS, heat (95-100°C for 5 minutes), and reducing agents

For Lower Molecular Weight Bands:

• Proteolytic degradation: Include complete protease inhibitor cocktails in lysis buffers

• Use freshly prepared samples or properly stored frozen samples to minimize degradation

• Consider reducing sample heating time if fragmentation is suspected

For Multiple Bands or Smears:

• Cross-reactivity: The antibody may recognize similar phospho-epitopes on related proteins

• Validate specificity using the phosphatase treatment control shown in the search results

• The alkaline phosphatase treatment should eliminate specific phospho-eIF4E bands

Validation Approaches:

• Compare patterns with total eIF4E antibodies to distinguish phospho-specific from general eIF4E bands

• Use samples from cells treated with EGF or serum as positive controls

• Compare with alternative phospho-eIF4E antibodies (the search results mention both EP2151Y monoclonal and a polyclonal antibody )

Technical Optimizations:

• Gradient gels can improve resolution of closely migrating bands

• Longer SDS-PAGE running times may better separate closely migrating species

• Optimization of transfer conditions for small proteins (25 kDa range) by adjusting methanol concentration or using specialized transfer buffers

A methodical approach to troubleshooting, combined with appropriate controls, will help identify the source of unexpected bands and guide protocol adjustments to achieve clean, specific detection of phosphorylated eIF4E.

How can I incorporate Phospho-EIF4E (S209) detection in high-throughput screening assays?

Incorporating Phospho-EIF4E (S209) detection into high-throughput screening (HTS) assays requires optimization of scalable, reliable detection methods:

TR-FRET Assay Implementation:

• The THUNDER™ and HTRF TR-FRET assays described in the search results are specifically designed for HTS applications

• These homogeneous assays require no wash steps, making them automation-friendly

• The search results demonstrate robust performance with Z'-factor values of 0.68, indicating excellent assay quality for screening

Assay Optimization for HTS:

Controls and Normalization:

• Positive controls: EGF-treated cells (3-10 nM for 30 minutes) show strong phospho-eIF4E signals

• Negative controls: Unstimulated cells or cells treated with CGP 57380 inhibitor

• Include control wells on each plate for normalization of plate-to-plate variation

Screening Workflow Considerations:

• Primary screen: Use the TR-FRET assay to identify compounds that modulate eIF4E phosphorylation

• Counter-screen: Test for effects on total eIF4E levels to distinguish specific phosphorylation modulators

• Secondary validation: Confirm hits using orthogonal methods like Western blotting with the recombinant monoclonal antibody

Data Analysis Approaches:

• Use the 665 nm/615 nm emission ratio for quantification

• Apply standard HTS statistical parameters (Z', signal/background ratio, coefficient of variation)

• Implement dose-response testing of primary hits to establish potency

• The search results demonstrate successful inhibition curves for CGP 57380 in both HEK293 and HeLa cells

This systematic approach enables efficient screening of compound libraries for modulators of eIF4E phosphorylation, with applications in both basic research and drug discovery.

How can I use Phospho-EIF4E (S209) antibodies to study translation regulation in disease models?

Phospho-EIF4E (S209) antibodies can be powerful tools for investigating translation regulation in disease models, with applications spanning multiple research areas:

Cancer Research Applications:

• Monitor eIF4E phosphorylation as a biomarker of mTOR and MAPK pathway activation in tumor samples

• Compare phosphorylation levels between normal and malignant tissues using immunohistochemistry (IHC-P)

• Evaluate eIF4E phosphorylation as a predictive marker for response to targeted therapies (e.g., mTOR inhibitors, MNK inhibitors)

Neurodegenerative Disease Models:

• The search results indicate reactivity with rat brain lysates , suggesting utility in neurological research

• Investigate translational control alterations in models of Alzheimer's, Parkinson's, or ALS

• Study stress-induced translational reprogramming via eIF4E phosphorylation

Multi-Modal Experimental Approaches:

Translational Research Strategies:

• Correlate phospho-eIF4E levels with disease progression or therapeutic response

• Use TR-FRET assays to screen compound libraries for novel modulators of eIF4E phosphorylation

• Develop combinatorial treatment approaches targeting both eIF4E phosphorylation and related pathways

Experimental Considerations:

• Select appropriate models that reflect the disease pathophysiology

• Include relevant stimuli or stressors that mirror disease conditions

• Consider temporal dynamics of phosphorylation in relation to disease progression

• Use multiple detection methods (Western blot, TR-FRET, immunostaining) for comprehensive analysis

By utilizing Phospho-EIF4E (S209) antibodies across these diverse applications, researchers can gain insights into the role of translational regulation in disease pathogenesis and identify potential therapeutic targets or biomarkers.

Future directions in Phospho-EIF4E (S209) research

The study of Phospho-EIF4E (S209) continues to evolve with several promising future directions:

Integration with multi-omics approaches: Combining phospho-eIF4E detection with transcriptomics, proteomics, and ribosome profiling will provide comprehensive insights into how this phosphorylation event selectively influences translation of specific mRNA subsets.

Single-cell analysis: Adapting phospho-eIF4E detection methods for single-cell resolution will reveal cell-to-cell heterogeneity in translational regulation, particularly relevant in complex tissues and tumors.

In vivo dynamics: Development of methods to monitor eIF4E phosphorylation in real-time in living systems will advance our understanding of its temporal regulation in development and disease progression.

Therapeutic targeting: As understanding of eIF4E phosphorylation in disease contexts grows, development of targeted therapeutics modulating this phosphorylation may provide novel treatment approaches for cancers and other conditions with dysregulated translation.

Structural and mechanistic insights: Further investigation of how S209 phosphorylation alters eIF4E function at the molecular level will clarify the mechanistic basis for selective translational effects.