EIF4B Antibody

What is EIF4B and what is its role in cellular function?

EIF4B (eukaryotic translation initiation factor 4B) is an RNA-binding protein essential for the binding of mRNA to the 43S pre-initiation complex during translation initiation. The complex consists of the 40S ribosomal subunit bound to a ternary complex of eIF2, GTP, and Met-tRNA stabilized by eIF3 . EIF4B contains an arginine-rich motif (ARM) in its carboxy-terminus that binds RNA non-specifically, while a canonical RNA motif (RMM) near the amino terminus binds specifically to 18S rRNA . This dual binding capability facilitates the binding of the 40S subunit to mRNA by serving as a bridge between 18S rRNA and mRNA. Additionally, EIF4B stimulates the ATPase and RNA helicase activity of eIF4E in conjunction with eIF4F, a heterotrimeric protein .

What are the common applications for EIF4B antibodies in research?

EIF4B antibodies can be utilized in multiple experimental approaches:

These applications enable researchers to detect, localize, and quantify EIF4B protein in various experimental systems .

How do the mTOR/PI3K and MAPK pathways converge to regulate EIF4B activity, and what are the implications for experimental design?

The regulation of EIF4B through phosphorylation represents a sophisticated convergence point of the mTOR/PI3K and MAPK signaling pathways. Research has revealed a biphasic pattern of EIF4B phosphorylation that researchers must consider when designing pathway inhibition experiments:

• Early Phase (3-15 minutes post-stimulation): Predominantly mediated by the MAPK pathway, particularly ERK1/2. This phase is sensitive to U0126 (MEK inhibitor) but resistant to rapamycin (mTOR inhibitor) .

• Late Phase (60-90 minutes post-stimulation): Primarily regulated by the PI3K/mTOR pathway. This phase is sensitive to rapamycin .

Time-course experiments have demonstrated that serum-induced MAPK phosphorylation occurs rapidly (detectable at 3 minutes, peaking at 5 minutes), while S6K phosphorylation (an mTOR pathway marker) appears later and remains sustained for longer periods . Critically, EIF4B phosphorylation begins before S6K activation, supporting the temporal regulation model .

When designing experiments to study EIF4B regulation, researchers should include:

• Multiple time points spanning early (3-15 min) and late (60-90 min) phases

• Parallel assessment of pathway activation markers (phospho-ERK1/2, phospho-S6K)

• Combined inhibitor treatments (U0126 + rapamycin) to completely abrogate EIF4B phosphorylation

Understanding this temporal regulation is essential for correctly interpreting drug inhibition studies targeting translation regulation mechanisms .

What are the key considerations when validating antibody specificity for EIF4B detection, and how can researchers address potential cross-reactivity issues?

Validating antibody specificity for EIF4B requires multiple complementary approaches to ensure reliable experimental outcomes:

• Molecular weight verification: Compare observed bands with the expected molecular weight (approximately 80 kDa for EIF4B, despite calculated 69 kDa weight due to post-translational modifications) . Any additional bands should be carefully investigated.

• Cross-species reactivity assessment: While many anti-EIF4B antibodies show reactivity with human, mouse, and rat EIF4B , sequence variations might affect epitope recognition. When working with less common species, researchers should:

  • Perform sequence alignment between the immunogen and target species EIF4B

  • Validate antibody performance with positive and negative controls from the target species

  • Consider using multiple antibodies targeting different epitopes

• Epitope-specific considerations: Different antibodies target distinct regions of EIF4B. For example, some antibodies specifically target the N-terminal region . When studying phosphorylation-dependent functions, researchers should select antibodies whose epitopes don't include or aren't masked by phosphorylation sites.

• Knock-down/knock-out validation: The gold standard for specificity confirmation is demonstrating signal reduction in knock-down/knock-out systems. Researchers should consider siRNA or CRISPR-based approaches to deplete EIF4B and confirm antibody specificity.

• Controls for post-translational modifications: When studying phosphorylated EIF4B, include controls with phosphatase treatment to confirm that observed bands are indeed phosphorylation-dependent.

By implementing these validation strategies, researchers can minimize the risk of misinterpreting results due to antibody cross-reactivity or non-specific binding .

How does EIF4B phosphorylation status affect its function in different cellular contexts, and what experimental approaches best capture these dynamics?

EIF4B phosphorylation represents a critical regulatory mechanism that integrates multiple signaling pathways to control translation initiation in context-specific ways:

• Serum vs. Insulin stimulation differences: Research has shown distinct phosphorylation patterns in response to different stimuli. For example, serum stimulation produces a rapamycin-resistant component of EIF4B Ser422 phosphorylation that is not observed with insulin stimulation . This differential regulation suggests stimulus-specific engagement of signaling pathways.

• Temporal dynamics and functional implications: EIF4B phosphorylation occurs in a biphasic pattern, with early phosphorylation (3-15 minutes) mediated primarily by ERK1/2 MAPK signaling, and later phosphorylation (60-90 minutes) controlled by the mTOR pathway . These temporal dynamics likely represent distinct functional states of EIF4B in the translation initiation process.

To effectively capture these dynamics, researchers should employ:

• Phospho-specific antibodies: Use antibodies specifically recognizing phosphorylated Ser422 and other key sites

• Pathway inhibitor time-course studies: Employ inhibitors (rapamycin, U0126) at various time points to dissect pathway contributions

• Multi-parameter analysis: Simultaneously assess EIF4B phosphorylation, pathway activation markers (phospho-ERK1/2, phospho-S6K), and translation initiation complex formation

• Polysome profiling: Correlate phosphorylation status with active translation by analyzing polysome association

• Mutational analysis: Generate phospho-mimetic and phospho-deficient EIF4B mutants to directly assess functional consequences

For experimental design, researchers should include multiple cell types and stimulation conditions to account for context-specific regulation. Additionally, assessment of EIF4B's interaction with other translation initiation factors (using co-immunoprecipitation) in different phosphorylation states provides crucial insights into the functional consequences of this regulation .

What are the optimal conditions for detecting EIF4B via Western blotting, and how can researchers troubleshoot common issues?

Successful Western blotting for EIF4B requires careful optimization of several parameters:

Recommended Protocol:

• Sample preparation:

  • Extract proteins using RIPA or NP-40 buffer supplemented with phosphatase inhibitors (critical for preserving phosphorylation status)

  • Load 20-30μg of total protein per lane

  • Heat samples at 95°C for 5 minutes in reducing conditions

• Gel selection and transfer:

  • Use 8-10% SDS-PAGE gels to achieve optimal resolution around 80 kDa (the observed molecular weight of EIF4B)

  • Transfer to PVDF membrane at 100V for 60-90 minutes in standard transfer buffer (25mM Tris, 192mM glycine, 20% methanol)

• Antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour

  • Incubate with primary antibody at dilutions of 1:1000-1:4000 overnight at 4°C

  • Wash extensively with TBST (3 × 10 minutes)

  • Incubate with HRP-conjugated secondary antibody at 1:5000-1:10000 for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) detection systems

  • Expected band: approximately 80 kDa

Common Issues and Troubleshooting:

For phospho-specific detection, researchers should use phospho-specific antibodies and properly maintain phosphorylation status throughout the procedure by including phosphatase inhibitors in all buffers .

What considerations are important when using EIF4B antibodies for immunohistochemistry and immunofluorescence applications?

Successful immunostaining for EIF4B requires careful attention to several technical parameters:

Recommended Protocol for IHC/IF:

• Sample preparation:

  • Fixation: 4% paraformaldehyde (PFA) for cells or formalin-fixed paraffin-embedded (FFPE) tissues

  • For FFPE tissues: Perform antigen retrieval with TE buffer pH 9.0 (primary recommendation) or citrate buffer pH 6.0 (alternative)

  • For cells: Permeabilize with 0.1-0.5% Triton X-100 for 10 minutes

• Blocking and antibody incubation:

  • Block with 1-5% normal serum (from secondary antibody host species) with 0.1% Triton X-100

  • Apply primary antibody at 1:200-1:1000 dilution

  • Incubate overnight at 4°C or 1-2 hours at room temperature

  • For IF: Use fluorophore-conjugated secondary antibodies at 1:500-1:1000

• Validated positive controls:

  • Cell lines: MCF-7, HeLa (especially sodium arsenite-treated)

  • Tissues: Human stomach tissue has shown reliable positive staining

Special Considerations:

• Subcellular localization interpretation: EIF4B is primarily cytoplasmic, reflecting its role in translation. Nuclear staining should be carefully validated as it might represent:

  • Specific regulatory events

  • Antibody cross-reactivity

  • Fixation artifacts

• Signal specificity assessment:

  • Include isotype controls

  • Consider peptide competition assays

  • When possible, include EIF4B-depleted samples as negative controls

• Multiplex staining considerations: When co-staining for EIF4B and its interacting partners or phosphorylation markers:

  • Ensure primary antibodies are from different host species

  • Carefully select fluorophores to avoid spectral overlap

  • Consider sequential staining for phospho-specific antibodies

• Quantification approaches:

  • Measure staining intensity using standardized exposure settings

  • Consider using digital image analysis software for unbiased quantification

  • For translational studies, develop consistent scoring criteria (e.g., H-score method)

By optimizing these parameters, researchers can achieve specific and reproducible detection of EIF4B in various tissue and cell samples .

How can researchers effectively use EIF4B antibodies to investigate pathway-specific phosphorylation in response to various stimuli and inhibitors?

Investigating EIF4B phosphorylation dynamics requires strategic experimental design that captures both temporal aspects and pathway-specific contributions:

Recommended Experimental Approach:

• Stimulation and inhibitor treatment design:

  • Stimuli selection: Use serum for dual pathway activation or insulin for predominantly mTOR/PI3K pathway activation

  • Time-course design: Include early (3-15 min) and late (60-90 min) time points to capture biphasic phosphorylation

  • Inhibitor strategy:

    • Pre-treat cells with rapamycin (100 nM) for mTOR inhibition

    • Pre-treat with U0126 (10 μM) for MEK/ERK inhibition

    • Use both inhibitors simultaneously for complete blockade

• Sample analysis protocol:

  • Collect whole cell lysates in buffer containing phosphatase inhibitors

  • Perform Western blot analysis using:

    • Phospho-specific antibodies for EIF4B (Ser422)

    • Pathway activation markers: phospho-ERK1/2 (Thr202/Tyr204), phospho-S6K (Thr389)

    • Total EIF4B antibodies to normalize for expression levels

• Data interpretation framework:

  • Early phosphorylation (3-15 min): Primarily MAPK-dependent (U0126-sensitive)

  • Late phosphorylation (60-90 min): Primarily mTOR-dependent (rapamycin-sensitive)

  • Rapamycin-resistant, U0126-sensitive components indicate MAPK pathway contribution

Advanced Analysis Methods:

• Quantitative phospho-proteomic analysis:

  • Employ mass spectrometry to identify all phosphorylation sites and their relative stoichiometry

  • Use SILAC or TMT labeling for comparative analysis across conditions

• Single-cell analysis approaches:

  • Flow cytometry with phospho-specific antibodies to assess population heterogeneity

  • Use recommended dilution of approximately 0.40 μg per 10^6 cells in a 100 μl suspension

  • Include appropriate controls for phospho-specificity

• Functional correlation experiments:

  • Pair phosphorylation analysis with polysome profiling or translation assays

  • Correlate EIF4B phosphorylation status with assembly of translation initiation complexes

By implementing these methodological approaches, researchers can effectively dissect the complex regulatory mechanisms controlling EIF4B phosphorylation and its functional consequences in translation regulation .

What are the best practices for storage and handling of EIF4B antibodies to maintain optimal performance over time?

Proper handling and storage of EIF4B antibodies is critical for maintaining their performance and extending their usable lifespan:

Recommended Storage Conditions:

• Temperature: Store antibodies at -20°C for long-term storage. Most EIF4B antibodies remain stable for at least one year when properly stored .

• Aliquoting: For antibodies supplied in liquid form with glycerol:

  • Aliquoting is generally unnecessary for -20°C storage of small volumes (e.g., 20μl sizes)

  • For larger volumes, create working aliquots to avoid repeated freeze-thaw cycles

• Buffer composition: Most EIF4B antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) . Some formulations may include 0.1% BSA as a stabilizer.

• Working solution handling:

  • Store diluted working solutions at 4°C for short-term use (typically up to one week)

  • For dilutions without preservatives, prepare fresh solutions for each experiment

Performance Maintenance Strategies:

• Freeze-thaw minimization: Limit freeze-thaw cycles to preserve antibody performance:

  • Allow antibodies to thaw completely at 4°C before use

  • Return to -20°C promptly after use

  • Consider adding glycerol (to 50% final) if not already present

• Contamination prevention:

  • Use sterile technique when handling antibody stocks

  • Avoid introducing foreign material with pipette tips

  • Consider adding sodium azide (0.02%) to working dilutions for bacteriostatic properties

• Performance monitoring:

  • Include positive controls (e.g., HeLa or K-562 cell lysates for Western blot) in each experiment

  • Document lot numbers and monitor for any performance changes

  • Consider validation experiments when using a new lot

• Troubleshooting decreased performance:

  • For precipitation, centrifuge at low speed and test supernatant

  • For reduced signal, try fresh aliquot or new lot

  • Verify storage conditions were maintained

By following these storage and handling guidelines, researchers can maximize antibody lifespan and ensure consistent experimental results over time .

How can researchers optimize EIF4B antibody-based co-immunoprecipitation protocols to study protein-protein interactions in the translation initiation complex?

Co-immunoprecipitation (Co-IP) using EIF4B antibodies can provide valuable insights into translation initiation complex dynamics, but requires careful optimization:

Recommended Co-IP Protocol:

• Cell lysis optimization:

  • Use gentle lysis buffers to preserve protein complexes:

    • NP-40 buffer (0.5% NP-40, 150mM NaCl, 50mM Tris-HCl pH 8.0)

    • Add protease and phosphatase inhibitors freshly

  • Perform lysis on ice with minimal mechanical disruption

  • Centrifuge at 12,000×g for 10 minutes at 4°C to clear lysates

• Antibody selection and binding:

  • Choose EIF4B antibodies validated for Co-IP applications

  • Recommended amounts: 2-5μg antibody per 500μg protein lysate

  • Pre-clearing: Incubate lysate with Protein G beads for 1 hour at 4°C

  • Antibody binding: Incubate cleared lysate with antibody overnight at 4°C

• Immunoprecipitation and washing:

  • Add Protein G beads and incubate for 2-4 hours at 4°C

  • Wash beads 4-5 times with lysis buffer containing reduced detergent

  • Perform final wash with PBS to remove detergent

  • Elute complexes with sample buffer or gentle elution buffer

• Analysis of precipitated complexes:

  • Analyze by Western blot for known EIF4B interacting partners

  • Probe for components of the translation initiation complex

  • Consider mass spectrometry for unbiased interaction profiling

Critical Optimization Variables:

Advanced Approaches:

• Sequential Co-IP: To identify specific subcomplexes, perform sequential immunoprecipitation with different antibodies.

• Stimulus-dependent interactions: Compare EIF4B interactions under different conditions:

  • Serum stimulation vs. starvation

  • With/without pathway inhibitors (rapamycin, U0126)

  • Under stress conditions

• Phosphorylation-dependent interactions: Use phospho-specific antibodies or phosphatase treatments to determine how phosphorylation affects EIF4B protein interactions.

By optimizing these parameters, researchers can effectively use EIF4B antibodies to characterize the dynamic protein interaction network in translation initiation complex assembly .

What are the most common sources of variability in EIF4B antibody experiments, and how can researchers control for these factors?

Successful EIF4B detection and quantification requires awareness of multiple sources of variability and implementation of appropriate controls:

Major Sources of Variability:

• Antibody-related factors:

  • Lot-to-lot variation in commercial antibodies

  • Specificity for different EIF4B epitopes

  • Cross-reactivity with related proteins

  • Recognition of specific post-translational modifications

• Sample-related factors:

  • Cell/tissue type differences in EIF4B expression and modification

  • Growth conditions affecting EIF4B phosphorylation status

  • Sample harvesting and processing methods

  • Preservation of phosphorylation status

• Technical factors:

  • Variation in protein extraction efficiency

  • Transfer efficiency in Western blotting

  • Antibody incubation conditions

  • Detection system sensitivity and dynamic range

Recommended Control Strategies:

Standardization Approaches:

• Quantitative Western blotting:

  • Use dilution series of recombinant EIF4B or standard lysates

  • Employ digital imaging systems with linear dynamic range

  • Report relative rather than absolute values

• Phosphorylation analysis:

  • Always normalize phospho-specific signals to total EIF4B

  • Include pathway activation markers (phospho-ERK1/2, phospho-S6K)

  • Consider phosphatase-treated controls

• Cross-platform validation:

  • Verify key findings with multiple antibodies targeting different epitopes

  • Validate with complementary techniques (mass spectrometry)

  • Consider orthogonal approaches to confirm functional significance

By implementing these control strategies, researchers can minimize variability and increase confidence in the interpretation of EIF4B experimental data .

How can researchers accurately interpret changes in EIF4B phosphorylation patterns in the context of translation regulation studies?

Interpreting EIF4B phosphorylation data requires consideration of multiple factors, including temporal dynamics, pathway crosstalk, and functional consequences:

Interpretation Framework:

• Temporal context assessment:

  • Early phosphorylation (3-15 min post-stimulation): Primarily MAPK-dependent, reflects initial translation activation

  • Late phosphorylation (60-90 min): Predominantly mTOR-dependent, associated with sustained translation

  • Consider time point selection when comparing across studies

• Pathway contribution analysis:

  • Rapamycin-sensitive component: mTOR/PI3K pathway contribution

  • U0126-sensitive component: MAPK pathway contribution

  • Rapamycin-resistant, U0126-sensitive component: MAPK-specific regulation

  • Dual inhibitor effects: Complete pathway integration

• Stimulus-specific patterns:

  • Serum stimulation: Biphasic pattern with contributions from both pathways

  • Insulin stimulation: Predominantly mTOR-dependent with minimal MAPK contribution

  • Context-dependent interpretation is essential

Functional Correlation Guidelines:

• Translation initiation complex formation:

  • Correlate EIF4B phosphorylation with eIF4F assembly

  • Assess interaction with eIF4A and helicase activity

  • Connect to 43S pre-initiation complex binding to mRNA

• Phospho-site specific functions:

  • Ser422 phosphorylation: Primary regulatory site with documented functional significance

  • Multiple phosphorylation sites may have distinct or cooperative effects

  • Consider site-specific antibodies for comprehensive analysis

• Cell type and context considerations:

  • Expression levels of pathway components may affect interpretation

  • Cell-type specific phosphorylation patterns may reflect different pathway activities

  • Growth conditions significantly impact baseline phosphorylation status

Integrated Analysis Approaches:

• Multi-parameter assessment:

  • Always analyze EIF4B phosphorylation alongside pathway activation markers

  • Include functional readouts of translation (polysome profiles, protein synthesis rates)

  • Consider parallel analysis of other translation factors (eIF4G, 4E-BP1)

• Quantitative analysis methods:

  • Calculate phospho-EIF4B/total EIF4B ratios

  • Perform kinetic analysis of phosphorylation rates

  • Consider computational modeling for pathway integration

By applying this interpretation framework, researchers can more accurately extract meaningful insights from EIF4B phosphorylation data and connect these observations to functional outcomes in translation regulation .

What emerging techniques are advancing our understanding of EIF4B function, and how might researchers incorporate these approaches?

The field of EIF4B research continues to evolve with several emerging techniques offering new insights into its function and regulation:

• CRISPR-based approaches:

  • CRISPR knock-out/knock-in: Generate complete EIF4B knockout cell lines or introduce tagged versions for tracking

  • Base editing: Create specific phospho-deficient or phospho-mimetic mutations at key sites

  • CRISPRi/CRISPRa: Modulate EIF4B expression levels without complete elimination

• Advanced imaging techniques:

  • Super-resolution microscopy: Visualize EIF4B localization within translation initiation complexes

  • Live-cell imaging with fluorescent tags: Track EIF4B dynamics in real-time

  • Proximity labeling (BioID, APEX): Identify spatial proteomics of EIF4B in intact cells

• Single-cell approaches:

  • Single-cell Western blot: Analyze cell-to-cell variability in EIF4B expression and phosphorylation

  • Single-cell RNA-seq with translation metrics: Connect EIF4B activity to translational outcomes

  • Mass cytometry: Simultaneously analyze multiple signaling pathways affecting EIF4B

• Structural biology advances:

  • Cryo-EM of initiation complexes: Visualize EIF4B in the context of translation initiation

  • Hydrogen-deuterium exchange mass spectrometry: Map conformational changes upon phosphorylation

  • Integrative structural modeling: Combine multiple data types for complete structural understanding

Researchers can incorporate these approaches by establishing collaborations with specialized laboratories, investing in new technological platforms, and designing experiments that leverage these methods to address long-standing questions about EIF4B function in translation initiation and regulation .