EIF4EBP3 Antibody

What is EIF4EBP3 and what is its role in translational regulation?

EIF4EBP3 (also known as 4E-BP3) is a member of the EIF4E-binding protein family that functions as a repressor of translation initiation. It regulates EIF4E activity by preventing its assembly into the eIF4F complex, which is crucial for cap-dependent translation . The mechanism involves a phosphorylation-dependent switch: in its hypophosphorylated state, EIF4EBP3 competes with EIF4G1/EIF4G3 and strongly binds to EIF4E, leading to translation repression. Conversely, when hyperphosphorylated, it dissociates from EIF4E, allowing interaction between EIF4G1/EIF4G3 and EIF4E and consequently initiating translation .

Beyond translation repression, EIF4EBP3 also inhibits EIF4E-mediated mRNA nuclear export, suggesting a multifaceted role in gene expression regulation . The protein is involved in several signaling pathways, including TGF-Beta and Prolactin Signaling, indicating its broader role in cellular processes . Understanding EIF4EBP3's function provides insights into how cells regulate protein synthesis in response to various stimuli, making it a significant target for research into translational control mechanisms.

How do EIF4EBP3 antibodies differ from antibodies targeting other 4E-BP family members?

EIF4EBP3 antibodies can be categorized into those specific for EIF4EBP3 alone and those that recognize multiple family members. This distinction is crucial for experimental design and data interpretation:

Specificity profiles:

• EIF4EBP3-specific antibodies: These target unique epitopes found only in EIF4EBP3, enabling selective study of this protein without cross-reactivity with EIF4EBP1 or EIF4EBP2 .

• Pan-specific antibodies: Some antibodies recognize conserved regions across multiple family members, such as the anti-eIF4EBP1 + eIF4EBP2 + eIF4EBP3 (phospho T45) antibody . These are valuable for studying shared regulatory mechanisms within the 4E-BP family.

Phosphorylation-state specificity:
EIF4EBP3 function is regulated through phosphorylation, and many available antibodies are phospho-specific, such as those targeting phosphorylated T45 . These enable researchers to distinguish between active and inactive forms of the protein, critical for understanding its dynamic regulation.

Species reactivity:
While most EIF4EBP3 antibodies are validated for human samples, orthologs exist in mouse, rat, bovine, zebrafish, chimpanzee, and chicken species . Researchers studying animal models must select antibodies with appropriate cross-reactivity for their experimental system.

When selecting between different 4E-BP family antibodies, researchers should consider the specific research question, required specificity, and whether detection of phosphorylated forms is essential for their experimental aims.

What controls should be included when using EIF4EBP3 antibodies?

Proper controls are essential for ensuring reliable and interpretable results when using EIF4EBP3 antibodies across various applications:

Positive controls:

• Cells or tissues known to express EIF4EBP3 at detectable levels

• Recombinant EIF4EBP3 protein for antibody validation

• Phosphorylation-inducing treatments (e.g., insulin stimulation) when using phospho-specific antibodies

Negative controls:

• Samples where EIF4EBP3 is absent or knocked down (siRNA or CRISPR)

• Secondary antibody-only controls to assess non-specific binding

• Dephosphorylation treatments (e.g., phosphatase treatment) when using phospho-specific antibodies

Specificity controls:

• Peptide competition assays to confirm antibody specificity

• Comparison with alternative antibodies targeting different epitopes

• Validation in multiple cell types/tissues to confirm consistent results

Loading and technical controls:

• Housekeeping proteins (e.g., GAPDH, β-actin) for Western blots

• Total protein staining (e.g., Ponceau S) to normalize for loading variability

• For phospho-specific detection, parallel blots with antibodies recognizing total EIF4EBP3 regardless of phosphorylation state

What are the optimal conditions for Western blot detection of EIF4EBP3?

Achieving high-quality Western blot results for EIF4EBP3 requires careful optimization of several parameters:

Sample preparation:

• Rapid extraction in the presence of phosphatase inhibitors is critical for preserving phosphorylation states

• Use of RIPA or NP-40 buffer supplemented with protease inhibitors

• Sample heating at 70°C for 10 minutes rather than 95°C to prevent aggregation of this small protein (10.9 kDa)

Gel electrophoresis:

• 15-20% polyacrylamide gels or gradient gels (4-20%) to resolve this low molecular weight protein

• Longer running times at lower voltage (80-100V) for better resolution of phosphorylated forms

• Consider using Phos-tag™ acrylamide for enhanced separation of phosphorylated species

Transfer conditions:

• PVDF membranes with 0.2 μm pore size (rather than 0.45 μm) to better retain small proteins

• Semi-dry transfer systems with 20% methanol in transfer buffer

• Lower voltage/amperage transfers (25V for 1.5 hours) to prevent protein pass-through

Antibody incubation:

• Primary antibody dilution typically between 1:500-1:2000, optimized for each specific antibody

• Overnight incubation at 4°C with gentle rocking for maximum sensitivity

• 5% BSA in TBST as blocking and antibody dilution buffer, particularly for phospho-specific antibodies

Detection systems:

• Enhanced chemiluminescence (ECL) with extended exposure times for low abundance detection

• Consideration of fluorescent secondary antibodies for multiplex detection and better quantification

Researchers should perform optimization experiments by systematically varying these conditions to determine the optimal protocol for their specific experimental system and antibody.

How can I distinguish between phosphorylated and non-phosphorylated forms of EIF4EBP3?

Differentiating phosphorylation states of EIF4EBP3 is crucial for understanding its functional status in translational regulation:

Phospho-specific antibodies:
Antibodies targeting specific phosphorylation sites, such as the anti-eIF4EBP1/2/3 (phospho T45) antibody, allow direct detection of phosphorylated forms . These should be used in parallel with antibodies recognizing total EIF4EBP3 regardless of phosphorylation state.

Mobility shift analysis:

• Phosphorylated EIF4EBP3 typically migrates more slowly on SDS-PAGE

• Multiple bands may represent different phosphorylation states

• Resolution can be enhanced using modified separation techniques:

  • Phos-tag™ acrylamide gels

  • Longer gel runs

  • Higher percentage acrylamide gels (15-20%)

Phosphatase treatment:
Treating samples with lambda phosphatase before Western blotting provides a negative control - the disappearance of bands or mobility shifts confirms phosphorylation status.

Two-dimensional gel electrophoresis:
Combining isoelectric focusing with SDS-PAGE can separate proteins based on both charge (affected by phosphorylation) and molecular weight.

Mass spectrometry:
For definitive phosphorylation site mapping, mass spectrometry following immunoprecipitation with EIF4EBP3 antibodies can identify specific modified residues and their stoichiometry.

These complementary approaches allow researchers to comprehensively characterize EIF4EBP3 phosphorylation states, which is essential for understanding its role in the phosphorylation-dependent switch between translation repression and activation .

What methodologies exist for studying EIF4EBP3 interactions with other proteins?

Investigating EIF4EBP3's protein interactions, particularly with EIF4E and components of the translational machinery, provides crucial insights into its regulatory functions:

Co-immunoprecipitation (Co-IP):

• Using EIF4EBP3 antibodies to pull down protein complexes, followed by Western blotting for interaction partners

• Can be performed in either direction (using antibodies against suspected binding partners)

• Requires careful buffer optimization to preserve native interactions

• Cross-linking prior to lysis can stabilize transient interactions

Proximity Ligation Assay (PLA):
This technique allows visualization of protein interactions in situ with subcellular resolution by combining antibody recognition with PCR amplification, providing quantitative data on EIF4EBP3 interactions within intact cells.

Bimolecular Fluorescence Complementation (BiFC):
By tagging EIF4EBP3 and potential interaction partners with complementary fragments of fluorescent proteins, researchers can visualize interactions when the fragments come together to form a functional fluorophore.

Pull-down assays with recombinant proteins:
Using purified recombinant EIF4EBP3 protein as bait to identify direct binding partners, with subsequent mass spectrometry analysis.

Surface Plasmon Resonance (SPR):
This technique measures binding kinetics and affinity constants between EIF4EBP3 and its interaction partners, distinguishing between high and low-affinity interactions.

Yeast two-hybrid screening:
While more suitable for discovering novel interactions, this system can validate and characterize EIF4EBP3 binding partners when combined with mutational analysis.

The choice of method depends on whether the goal is to discover novel interactions, validate suspected interactions, or characterize the dynamics and regulation of known interactions. For studying phosphorylation-dependent interactions, such as those between EIF4EBP3 and EIF4E, it's essential to preserve phosphorylation states during experimental procedures .

How can I quantify changes in EIF4EBP3 phosphorylation in response to treatments?

Accurately measuring EIF4EBP3 phosphorylation dynamics is essential for understanding its regulation in response to stimuli:

Western blot quantification:

• Use phospho-specific antibodies such as anti-eIF4EBP1/2/3 (phospho T45)

• Always normalize phospho-signal to total EIF4EBP3 levels

• Implement technical replicates (minimum of three)

• Use fluorescent secondary antibodies for wider linear detection range

• Densitometric analysis with appropriate software (ImageJ, Image Studio Lite)

Quantitative phosphoproteomics:

• SILAC (Stable Isotope Labeling with Amino acids in Cell culture) or TMT (Tandem Mass Tag) labeling

• Phosphopeptide enrichment using TiO₂ or IMAC (Immobilized Metal Affinity Chromatography)

• Mass spectrometry analysis to identify and quantify multiple phosphorylation sites simultaneously

• Allows discovery of novel phosphorylation sites beyond T45

ELISA-based quantification:

• Sandwich ELISA with capture antibodies against total EIF4EBP3 and detection antibodies against phospho-epitopes

• Provides high-throughput capability for multiple samples

• Higher sensitivity than Western blotting for samples with low EIF4EBP3 expression

In-cell Western/ICW assay:

• Allows direct measurement in fixed cells

• Dual-channel detection of phospho and total protein

• Suitable for high-throughput screening of compounds affecting EIF4EBP3 phosphorylation

Kinase assays:

• In vitro kinase assays with purified components to measure direct phosphorylation

• Particularly useful for characterizing specific kinases responsible for EIF4EBP3 modification

When reporting quantitative phosphorylation data, it's essential to include appropriate statistical analysis and clearly state the normalization method used. Time-course experiments can provide valuable insights into the kinetics of phosphorylation changes, which may reveal important aspects of signaling pathway dynamics affecting EIF4EBP3 function.

What are common challenges in detecting endogenous EIF4EBP3 and how can they be addressed?

Detecting endogenous EIF4EBP3 presents several technical challenges that researchers should anticipate and address:

Low expression levels:

• EIF4EBP3 is often expressed at lower levels than EIF4EBP1 and EIF4EBP2

• Solution: Use more sensitive detection methods such as enhanced chemiluminescence (ECL) substrate or signal amplification systems

• Increase protein loading (50-100 μg total protein versus standard 20-30 μg)

• Consider concentration steps such as immunoprecipitation before Western blotting

Cross-reactivity with other 4E-BP family members:

• High sequence homology with EIF4EBP1 and EIF4EBP2 can lead to non-specific detection

• Solution: Validate antibody specificity using recombinant proteins or knockout/knockdown controls

• Use antibodies targeting unique regions of EIF4EBP3

• Consider using pan-specific antibodies when appropriate, with careful interpretation

Small protein size (10.9 kDa):

• Proteins of this size can easily transfer through membranes or diffuse from gels

• Solution: Use 0.2 μm pore PVDF membranes and optimize transfer conditions

• Consider fixation of proteins in gel with 0.4% glutaraldehyde before transfer

• Use high percentage (15-20%) acrylamide gels for better resolution

Phosphorylation heterogeneity:

• Multiple phosphorylation states can complicate band patterns

• Solution: Use phosphatase treatment controls to identify all forms

• Employ Phos-tag™ gels for better separation of phosphorylated species

• Use a combination of phospho-specific and total protein antibodies

Antibody sensitivity and specificity:

• Variable quality among commercial antibodies

• Solution: Validate with multiple antibodies targeting different epitopes

• Test multiple antibody dilutions and incubation conditions

• Consider using recombinant antibody formats for better reproducibility

By anticipating these challenges and implementing the suggested solutions, researchers can significantly improve their ability to detect and study endogenous EIF4EBP3 across different experimental systems.

How should I optimize immunohistochemistry protocols for EIF4EBP3 detection in tissue samples?

Successful immunohistochemical detection of EIF4EBP3 in tissue sections requires careful optimization of multiple steps:

Tissue fixation and processing:

• Optimal fixation with 10% neutral buffered formalin for 24-48 hours

• Avoid prolonged fixation which can mask epitopes

• Consider using freshly cut sections from paraffin blocks (within 1 week) for optimal antigenicity

Antigen retrieval methods:

• Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• For phospho-epitopes, EDTA buffer (pH 8.0) often yields better results

• Optimize retrieval time (typically 15-20 minutes) and temperature (95-100°C)

Blocking conditions:

• Use 5-10% normal serum from the same species as the secondary antibody

• Add 0.1-0.3% Triton X-100 for improved penetration in thicker sections

• Consider dual blocking with serum and 1% BSA to reduce background

Primary antibody optimization:

• Test multiple dilutions (typically starting at 1:100 and ranging to 1:1000)

• Optimize incubation time and temperature (overnight at 4°C often yields best results)

• For phospho-specific antibodies, include phosphatase inhibitors in all buffers

Detection systems:

• Polymer-based detection systems often provide better sensitivity than standard ABC methods

• For low abundance targets, consider tyramide signal amplification (TSA) systems

• Use DAB or AEC chromogens based on desired signal intensity and counterstaining

Controls and validation:

• Include positive control tissues with known EIF4EBP3 expression

• Use negative controls (primary antibody omission and isotype controls)

• Consider dual staining with another antibody to confirm cell type-specific expression

Counterstaining and mounting:

• Light hematoxylin counterstaining to avoid obscuring specific signals

• Aqueous mounting media for AEC and permanent mounting for DAB

A systematic approach to optimizing each of these parameters will maximize the likelihood of successful EIF4EBP3 detection in tissue samples while minimizing background and non-specific staining.

How can I address non-specific binding and background issues with EIF4EBP3 antibodies?

Non-specific binding and high background are common challenges when working with EIF4EBP3 antibodies. Here are methodological approaches to troubleshoot and resolve these issues:

For Western blotting:

• Increase blocking time and concentration (5% BSA or 5% non-fat dry milk for 1-2 hours)

• Include 0.1-0.3% Tween-20 in washing buffers with increased washing frequency and duration

• Optimize primary antibody dilution through titration experiments (1:500 to 1:5000)

• Reduce secondary antibody concentration (typically 1:10,000 to 1:20,000)

• Pre-adsorb antibodies against lysates from cells lacking EIF4EBP3 expression

• Use membrane stripping protocols with validation to confirm complete removal of previous antibodies

For immunohistochemistry/immunofluorescence:

• Implement dual blocking strategy (protein block followed by serum block)

• Use fragment antigen-binding (Fab) secondary antibodies to reduce Fc receptor binding

• Include additional washing steps with PBS containing 0.05-0.1% Tween-20

• Treat sections with 0.3% hydrogen peroxide before antibody incubation to quench endogenous peroxidase

• Consider autofluorescence quenching for immunofluorescence applications

Antibody validation strategies:

• Perform peptide competition assays to confirm specificity

• Test antibody on EIF4EBP3 knockdown/knockout samples as negative controls

• Compare staining patterns across multiple antibodies targeting different epitopes

• Use recombinant monoclonal antibodies for improved consistency

Sample-specific considerations:

• For tissues with high endogenous biotin, use biotin-free detection systems

• In highly vascularized tissues, block endogenous immunoglobulins with F(ab) fragments

• For tissues with high background, consider reducing primary antibody incubation time

• Use detergent optimization to reduce membrane-associated non-specific binding

Systematic application of these approaches, with careful documentation of optimization steps, will help researchers identify the specific sources of background and non-specific binding for their particular experimental system.

What factors affect the reproducibility of EIF4EBP3 antibody-based experiments?

Ensuring reproducible results with EIF4EBP3 antibodies requires attention to several critical factors:

Antibody quality and consistency:

• Lot-to-lot variation in polyclonal antibodies can significantly impact results

• Solution: Use recombinant monoclonal antibodies when available

• Record lot numbers and maintain consistency throughout a study

• Perform validation with each new lot using positive and negative controls

Sample preparation variables:

• Cell culture conditions (confluence, passage number, media composition)

• Lysis buffer composition and extraction protocols

• Phosphorylation state preservation (phosphatase inhibitors, sample handling time)

• Protein quantification method and loading accuracy

Experimental conditions standardization:

• Consistent blocking reagents and incubation times

• Temperature control during all incubation steps

• Buffer preparation and pH verification

• Instrument settings for detection and imaging

Data analysis consistency:

• Standardized quantification methods

• Appropriate normalization controls

• Consistent image acquisition settings

• Blinding during analysis when possible

Documentation and reporting:

Biological variables:

• Cell type-specific expression patterns of EIF4EBP3

• Response variation to treatments across cell lines

• Temporal dynamics of phosphorylation changes

• Influence of cell cycle stage on EIF4EBP3 levels and modification

By systematically controlling these variables and maintaining detailed records of experimental conditions, researchers can significantly improve the reproducibility of their EIF4EBP3 antibody-based experiments and ensure reliable data for publication and further research.

How are EIF4EBP3 antibodies used to study mTOR signaling pathways?

EIF4EBP3, as a downstream effector of mTOR signaling, provides a valuable readout for pathway activity. Antibodies targeting this protein enable researchers to investigate this critical signaling cascade:

Pathway activation monitoring:

• Phospho-specific antibodies targeting residues like T45 allow assessment of mTOR activity

• These can be used to examine temporal dynamics of pathway activation following stimuli

• Comparative analysis with other mTOR substrates (e.g., S6K1) provides insight into pathway branching

Pharmacological intervention studies:

• EIF4EBP3 phosphorylation status can be used to evaluate efficacy of mTOR inhibitors

• Dose-response relationships can be established using quantitative Western blotting

• Antibodies enable assessment of pathway reactivation mechanisms in drug resistance

Nutrient sensing mechanisms:

• Changes in EIF4EBP3 phosphorylation in response to amino acid availability

• Glucose deprivation effects on mTOR-mediated translational control

• Oxygen sensing and hypoxic response pathways intersecting with mTOR

Methodological approaches:

• Multi-parameter flow cytometry with phospho-specific antibodies for single-cell analysis

• Immunoprecipitation followed by mass spectrometry to identify novel interacting partners

• High-content imaging to assess subcellular localization changes following mTOR modulation

Integration with other techniques:

• Combination with polysome profiling to correlate phosphorylation status with translation efficiency

• CRISPR-mediated generation of phospho-mutant EIF4EBP3 to dissect specific contributions of individual sites

• Proximity-based labeling methods (BioID, APEX) to map interaction networks in different activation states

By leveraging EIF4EBP3 antibodies in these diverse applications, researchers can gain mechanistic insights into mTOR signaling with implications for understanding diseases characterized by dysregulated translational control.

What role does EIF4EBP3 play in disease mechanisms, and how are antibodies contributing to this research?

EIF4EBP3 has emerging roles in several disease processes, with antibody-based research providing critical insights:

Cancer biology:

• Dysregulated cap-dependent translation is a hallmark of many cancers

• EIF4EBP3 antibodies enable assessment of translational control mechanisms in tumor samples

• Phosphorylation status correlates with mTOR pathway activation and therapeutic responses

• Research has identified associations with thymus lymphoma specifically

Neurodegenerative diseases:

• Aberrant protein synthesis contributes to neurodegeneration pathology

• Antibody-based studies reveal altered EIF4EBP3 regulation in disease models

• Comparisons with other 4E-BP family members help distinguish unique contributions

• Phosphorylation patterns may serve as biomarkers for disease progression

Metabolic disorders:

• EIF4EBP3 functions in insulin signaling pathways affecting protein synthesis

• Antibodies enable tissue-specific analysis of translational control in metabolic tissues

• Differential regulation of EIF4EBP3 versus other family members in diabetes models

Methodological approaches in disease research:

• Tissue microarray analysis with phospho-specific antibodies in patient samples

• Correlation of EIF4EBP3 status with clinical outcomes and treatment responses

• Multiplexed immunohistochemistry to assess pathway activation in heterogeneous tissues

• Single-cell analysis to identify cell type-specific dysregulation

Therapeutic development:

• EIF4EBP3 antibodies in high-throughput screens for compounds affecting mTOR signaling

• Monitoring phosphorylation as pharmacodynamic markers for targeted therapies

• Development of phospho-specific antibodies as diagnostic tools

This research is expanding our understanding of how translational control mechanisms contribute to disease pathogenesis and revealing potential opportunities for therapeutic intervention targeting the EIF4E/4E-BP regulatory axis.

What are emerging techniques for studying EIF4EBP3 function beyond traditional antibody applications?

While antibodies remain fundamental tools, innovative approaches are expanding our ability to study EIF4EBP3:

CRISPR-based approaches:

• Gene editing to create endogenous tagged versions of EIF4EBP3

• Knock-in of phospho-mimetic or phospho-deficient mutations at key regulatory sites

• CRISPRi/CRISPRa for modulating expression levels without complete knockout

• Base editing for introducing specific mutations to regulatory phosphorylation sites

Proximity labeling proteomics:

• BioID or TurboID fusion proteins to identify proximal interacting partners

• APEX2-based approaches for temporal mapping of interaction networks

• Integration with quantitative proteomics to assess dynamic changes in interactome

Live cell imaging techniques:

• FRET biosensors to monitor EIF4EBP3-EIF4E interactions in real-time

• Optogenetic control of EIF4EBP3 phosphorylation or localization

• Fluorescent translational reporters to assess functional consequences of EIF4EBP3 manipulation

Single-cell analyses:

• scRNA-seq combined with proteomics to correlate EIF4EBP3 protein levels with transcriptome

• Mass cytometry (CyTOF) with metal-conjugated antibodies for multi-parameter analysis

• Spatial transcriptomics to map EIF4EBP3 activity in tissue contexts

Structural biology advances:

• Cryo-EM studies of EIF4EBP3 in complex with translational machinery

• Hydrogen-deuterium exchange mass spectrometry to probe conformational changes

• NMR studies of phosphorylation-induced structural alterations

Systems biology integration:

• Multi-omics approaches combining transcriptomics, proteomics, and translatomics

• Mathematical modeling of EIF4EBP3 in translational control networks

• Machine learning to predict functional consequences of EIF4EBP3 modifications

These emerging technologies are complementary to traditional antibody-based methods and are enabling more comprehensive understanding of EIF4EBP3 biology in normal and disease states. Integration of multiple approaches provides the most robust insights into this key regulator of translation.

How can EIF4EBP3 antibodies be used in multi-parameter analyses of translational control?

Modern translational research increasingly employs multi-parameter approaches to understand complex regulatory networks involving EIF4EBP3:

Multiplexed immunofluorescence:

• Simultaneous detection of EIF4EBP3 with other translation factors

• Co-localization analysis with RNA granules, stress granules, or P-bodies

• Quantitative image analysis across multiple cell states or treatments

• Spatial relationship with upstream kinases and downstream effectors

Flow cytometry and mass cytometry:

• Multi-parameter analysis combining EIF4EBP3 phosphorylation with cell cycle markers

• Integration with readouts of global translation rates (e.g., puromycin incorporation)

• Single-cell correlation of EIF4EBP3 status with activation of parallel signaling pathways

• Rare cell population identification based on unique EIF4EBP3 regulatory patterns

Proteomics integration:

• Phospho-proteomics to place EIF4EBP3 modification in context of global signaling events

• Correlation with changes in translatome using techniques like polysome profiling

• Quantitative analysis of EIF4EBP3 binding partners under different conditions

• Pathway analysis to identify feedback mechanisms and regulatory circuits

Multi-omics experimental designs:

Data integration approaches:

• Computational models incorporating EIF4EBP3 phosphorylation state with translation rates

• Network analysis to identify regulatory hubs and feedback mechanisms

• Machine learning to predict translational outcomes based on EIF4EBP3 status

• Visualization tools for multi-dimensional data exploration

These multi-parameter approaches provide a systems-level understanding of how EIF4EBP3 functions within the broader context of translational control networks, moving beyond isolated protein studies to comprehend its role in integrated cellular processes.