EIF4EBP1 Antibody, HRP conjugated

What is EIF4EBP1 and what cellular functions does it regulate?

EIF4EBP1 (Eukaryotic translation initiation factor 4E-binding protein 1), also known as 4E-BP1 or PHAS-I, functions as a critical repressor of translation initiation. This protein regulates EIF4E activity by preventing its assembly into the eIF4F complex through competitive binding. In its hypophosphorylated state, EIF4EBP1 competes with EIF4G1/EIF4G3 and strongly binds to EIF4E, effectively repressing translation. Conversely, when hyperphosphorylated, EIF4EBP1 dissociates from EIF4E, permitting interaction between EIF4G1/EIF4G3 and EIF4E, thus initiating translation. EIF4EBP1 serves as a key mediator in the regulation of protein translation in response to hormones, growth factors, and other stimuli, primarily functioning through the MAP kinase and mTORC1 signaling pathways .

How do different phosphorylation states of EIF4EBP1 affect its biological activity?

The biological activity of EIF4EBP1 is primarily regulated through its phosphorylation status at multiple sites. Research indicates that EIF4EBP1 undergoes dynamic and spatially regulated patterns of phosphorylation at specific residues that determine its association with EIF4E and consequently its ability to repress translation. Notably, phosphorylation at Thr37, Thr46, Ser64, and Thr69 has been extensively studied. When EIF4EBP1 remains hypophosphorylated, it strongly binds to EIF4E and represses translation. Progressive phosphorylation at these key residues reduces its affinity for EIF4E, ultimately allowing translation initiation to proceed. These phosphorylation events appear to be regulated in spatially specific ways, particularly in relation to spindle formation and cell cycle progression in oocytes, suggesting sophisticated coordination of localized translation with specific cellular processes .

Why is EIF4EBP1 significant in cancer research, particularly regarding treatment resistance?

EIF4EBP1 has emerged as a crucial factor in cancer biology, particularly in the development of treatment resistance. Recent studies have shown that EIF4EBP1 is significantly upregulated in breast cancer (BC) tissues and tamoxifen (TAM)-resistant cell lines. High expression of EIF4EBP1 has been strongly correlated with increased cell proliferation, invasion, migration, and notably, the development of tamoxifen resistance in breast cancer cells. Gene Set Enrichment Analysis (GSEA) has further identified that EIF4EBP1 potentially influences several critical pathways in cancer progression and treatment response, including the Hedgehog signaling pathway, PPAR signaling pathway, and ErbB signaling pathway. Particularly noteworthy is the finding that knocking down EIF4EBP1 can reverse tamoxifen resistance, while its overexpression promotes resistance, suggesting its potential as both a prognostic marker and therapeutic target in cancer treatment .

What are the optimal protocols for using HRP-conjugated EIF4EBP1 antibodies in Western blotting?

For optimal Western blotting results with HRP-conjugated EIF4EBP1 antibodies, begin with proper sample preparation by extracting proteins in a buffer containing phosphatase inhibitors to preserve phosphorylation states. Load 20-30 μg of protein per lane on 12-15% SDS-PAGE gels to adequately resolve the relatively small EIF4EBP1 protein (approximately 15-20 kDa). After transfer to PVDF membranes (preferred over nitrocellulose for phosphorylated proteins), block in 5% non-fat milk or BSA in TBST for 1 hour at room temperature. For HRP-conjugated primary antibodies, dilute according to manufacturer specifications (typically 1:1000 to 1:5000) and incubate overnight at 4°C. Since the antibody is already HRP-conjugated, no secondary antibody is required. Wash extensively with TBST (at least 3×10 minutes) before detection with ECL substrate. Note that EIF4EBP1 can appear as multiple bands representing different phosphorylation states (α, β, and γ isoforms), so proper resolution is crucial for distinguishing these states .

How can I optimize immunohistochemistry protocols for both total and phospho-specific EIF4EBP1 antibodies?

For effective immunohistochemistry with EIF4EBP1 antibodies, tissue preparation is critical. Use either formalin-fixed, paraffin-embedded (FFPE) sections (4-6 μm thick) or frozen sections as appropriate. For FFPE tissues, complete antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0) heated to 95-100°C for 15-20 minutes, as this significantly improves antibody access to EIF4EBP1 epitopes, especially for phospho-specific antibodies. Block endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes if using HRP-conjugated antibodies. For blocking, use 5-10% normal serum from the same species as the secondary antibody (if using an unconjugated primary). Dilute antibodies appropriately (typically 1:100 to 1:500 for IHC applications) and incubate overnight at 4°C in a humidified chamber. For phospho-specific antibodies, it's essential to include phosphatase inhibitors in all buffers to preserve phosphorylation states. When using HRP-conjugated antibodies directly, proceed to detection with DAB substrate after thorough washing. Include appropriate positive control tissues and use sequential sections with phospho-specific and total EIF4EBP1 antibodies for comparative analysis of phosphorylation status .

What methodological approaches can help distinguish between different phosphorylation states of EIF4EBP1?

To effectively distinguish between different phosphorylation states of EIF4EBP1, employ a multi-faceted methodological approach. First, use phospho-site specific antibodies that recognize distinct phosphorylation sites (Thr37, Thr46, Ser64, Thr69) in parallel with antibodies recognizing total EIF4EBP1. For Western blotting, take advantage of the mobility shift that occurs with increased phosphorylation – hypophosphorylated forms migrate faster (α band), while hyperphosphorylated forms migrate more slowly (γ band), with intermediately phosphorylated forms appearing as the β band. Combining these approaches provides a comprehensive phosphorylation profile. For more precise quantification, consider using Phos-tag SDS-PAGE, which can separate proteins based on their phosphorylation status with greater resolution than standard SDS-PAGE. For spatial localization of different phosphorylation states, employ dual immunofluorescence using combinations of phospho-specific antibodies with distinct fluorophores. When analyzing data, calculate phosphorylation ratios (phospho-specific signal divided by total protein signal) to normalize for variations in total protein expression. This integrated approach allows for nuanced analysis of EIF4EBP1's phosphorylation state, which is crucial for understanding its role in translation regulation .

How can EIF4EBP1 antibodies be utilized to study localized translation in cellular compartments?

To investigate localized translation using EIF4EBP1 antibodies, implement cellular compartment-specific analyses through advanced microscopy techniques. Begin with high-resolution confocal microscopy using phospho-specific EIF4EBP1 antibodies to identify distinct subcellular localization patterns of differently phosphorylated forms. This approach has revealed that phosphorylated EIF4EBP1 variants appear at different positions along the spindle-chromosome complex (SCC) at specific times during meiotic maturation, suggesting spatially regulated translation control. For more detailed spatial analysis, employ super-resolution microscopy techniques such as STORM or PALM. Combine EIF4EBP1 immunostaining with fluorescent in situ hybridization (FISH) to simultaneously visualize EIF4EBP1 phosphorylation status and specific mRNAs, which can reveal co-localization patterns indicative of translation regulation zones. For dynamic studies, use live-cell imaging with fluorescently tagged EIF4EBP1 constructs paired with subsequent immunostaining validation using phospho-specific antibodies. Subcellular fractionation followed by Western blotting with phospho-specific antibodies can provide biochemical confirmation of compartment-specific EIF4EBP1 phosphorylation states. This multi-technique approach has revealed that spatially restricted patterns of EIF4EBP1 phosphorylation likely promote localized translation within mammalian oocytes, contributing to critical processes like spindle formation, maintenance, and chromosome segregation .

What experimental design strategies are essential when investigating EIF4EBP1's role in drug resistance mechanisms?

When investigating EIF4EBP1's role in drug resistance mechanisms, implement a comprehensive experimental design that begins with establishing resistant cell lines through progressive drug exposure, as demonstrated in studies of tamoxifen-resistant breast cancer. Compare expression levels of total and phosphorylated EIF4EBP1 between parental and resistant cells using Western blotting and quantitative PCR. Following this baseline characterization, employ genetic manipulation approaches including siRNA-mediated knockdown and CRISPR/Cas9 gene editing to modulate EIF4EBP1 levels, then assess changes in drug sensitivity using viability assays (e.g., CCK-8) and colony formation assays. For mechanistic insights, analyze pathway alterations through phosphorylation status of downstream targets. Incorporate RNA-seq and proteomics to identify associated gene expression changes and potential compensatory mechanisms. Validate findings through rescue experiments where EIF4EBP1 is re-expressed in knockout cells. For clinical relevance, analyze patient-derived samples to correlate EIF4EBP1 expression with treatment response and employ xenograft models to confirm in vitro findings. This multi-layered approach has proven effective in demonstrating that EIF4EBP1 overexpression promotes tamoxifen resistance in breast cancer, while its knockdown can restore drug sensitivity .

How can researchers effectively use EIF4EBP1 antibodies to investigate the coordination between cell cycle progression and mRNA translation?

To investigate the coordination between cell cycle progression and mRNA translation using EIF4EBP1 antibodies, implement synchronized cell systems to capture distinct cell cycle phases. Use double thymidine block or nocodazole treatment for synchronization, then collect cells at specific timepoints for analysis. Employ Western blotting with phospho-specific EIF4EBP1 antibodies (targeting Thr37/46, Ser64, and Thr69) alongside cell cycle markers (cyclin B1, phospho-histone H3) to correlate EIF4EBP1 phosphorylation states with cell cycle progression. For spatial coordination, perform immunofluorescence co-staining of phospho-EIF4EBP1 with spindle markers (α-tubulin) and chromosomal markers (DAPI) to visualize localization patterns during different mitotic or meiotic stages. Combine these approaches with polysome profiling to directly assess translation efficiency of specific mRNAs across the cell cycle. For mechanistic insights, use selective inhibitors of mTOR (rapamycin, torin) and cell cycle regulators (CDK inhibitors) to perturb the system, then analyze effects on EIF4EBP1 phosphorylation and localized translation. This integrated approach has revealed that dynamic spatially-restricted patterns of EIF4EBP1 phosphorylation promote localized translation that coordinates spindle formation and maintenance with cell cycle progression, a mechanism conserved across cell types and evolutionarily conserved between mammals and other vertebrates .

What are the most common technical challenges with phospho-specific EIF4EBP1 antibodies and how can they be addressed?

The most common technical challenges with phospho-specific EIF4EBP1 antibodies include phosphorylation preservation, specificity issues, and variable sensitivity. To address phosphorylation preservation challenges, immediately add phosphatase inhibitor cocktails to all buffers during sample preparation and maintain samples at 4°C throughout processing. For cell lysis, use a buffer containing sodium fluoride (50 mM), sodium pyrophosphate (10 mM), sodium orthovanadate (1 mM), and β-glycerophosphate (10 mM). For tissue samples, snap-freeze immediately after collection and process without thawing cycles. Regarding specificity concerns, validate antibodies using positive controls (insulin-stimulated samples for increased phosphorylation) and negative controls (samples treated with phosphatase or mTOR inhibitors like rapamycin). For challenging phospho-sites, consider using Phos-tag SDS-PAGE to separate proteins based on phosphorylation status before Western blotting. To address sensitivity issues, optimize blocking conditions (5% BSA is often superior to milk for phospho-epitopes) and primary antibody concentration through titration experiments. Extended primary antibody incubation (overnight at 4°C) and signal amplification systems like tyramide signal amplification for immunohistochemistry can enhance detection of low-abundance phosphorylation sites. For consistent results, aliquot antibodies to avoid freeze-thaw cycles and prepare all samples identically with standardized protein amounts .

What validation methods should be employed to ensure the specificity and reliability of EIF4EBP1 antibody results?

To ensure specificity and reliability of EIF4EBP1 antibody results, implement a comprehensive validation strategy beginning with positive and negative controls. Use lysates from cell lines with known EIF4EBP1 expression profiles as positive controls, and include EIF4EBP1 knockout or knockdown samples as negative controls. For phospho-specific antibodies, treat samples with lambda phosphatase to confirm signal loss, and use mTOR inhibitors (rapamycin, Torin1) to reduce phosphorylation at specific sites. Peptide competition assays, where pre-incubation of the antibody with the immunizing peptide blocks specific binding, provide another specificity check. Cross-validate results using multiple antibodies targeting different epitopes of EIF4EBP1 from different suppliers. For rigorous validation, perform immunoprecipitation followed by mass spectrometry to confirm that the antibody is pulling down the correct protein. When testing new applications, compare results with established literature findings for consistency. Document batch-to-batch variation by maintaining reference samples that can be used to normalize signal intensity across experiments. Finally, confirm specificity in your particular experimental system by showing that manipulation of EIF4EBP1 levels (overexpression or knockdown) produces corresponding changes in antibody signal. This multi-faceted validation approach ensures that experimental findings accurately reflect EIF4EBP1 biology rather than antibody artifacts .

How can researchers optimize signal-to-noise ratio when using HRP-conjugated EIF4EBP1 antibodies in various applications?

To optimize signal-to-noise ratio when using HRP-conjugated EIF4EBP1 antibodies, implement application-specific strategies. For Western blotting, begin with thorough membrane blocking using 5% BSA in TBST rather than milk, as milk contains phosphoproteins that may increase background with phospho-specific antibodies. Optimize primary antibody dilution through careful titration experiments (typically starting with 1:1000-1:5000 dilutions for HRP-conjugated antibodies). Extend washing steps to 5-6 washes of 10 minutes each with fresh TBST buffer to remove non-specifically bound antibodies. For immunohistochemistry applications, perform meticulous blocking of endogenous peroxidase activity using 3% hydrogen peroxide for 10-15 minutes before antibody application, followed by thorough washing. Include an avidin-biotin blocking step if using detection systems that incorporate these molecules. Optimize antigen retrieval conditions specifically for EIF4EBP1 epitopes, testing both citrate and EDTA-based buffers at different pH values. For all applications, reduce non-specific binding by including 0.1-0.3% Triton X-100 in blocking buffers. When developing the signal, use freshly prepared ECL substrate and optimize exposure times to avoid signal saturation while maintaining sensitivity. For challenging samples with high background, consider signal enhancement systems or switch to fluorescent detection methods with directly labeled primary antibodies for improved signal discrimination .

What analytical approaches should be used to quantify EIF4EBP1 phosphorylation in relation to total protein levels?

For rigorous quantification of EIF4EBP1 phosphorylation relative to total protein levels, implement a multi-step analytical approach. Begin by capturing Western blot images within the linear dynamic range of detection using a digital imaging system rather than film. Use biological replicates (minimum n=3) and technical duplicates to ensure statistical validity. For data analysis, employ densitometry software (ImageJ, Image Lab) to quantify band intensities, ensuring background subtraction is consistent across all samples. Calculate the phospho-to-total ratio by dividing the signal intensity of phospho-specific antibody bands by that of total EIF4EBP1 bands from the same sample, preferably run on parallel gels loaded with equal amounts of the same lysate. This normalization controls for variations in total protein expression across samples. For comprehensive phosphorylation profiles, analyze all major phosphorylation sites (Thr37/46, Ser64, Thr69) simultaneously. Present data as fold change relative to control conditions, using appropriate statistical tests (t-test, ANOVA) to determine significance. For higher precision in complex samples, consider using Phos-tag SDS-PAGE to resolve and quantify multiple phosphorylation states simultaneously. When multiple bands are present (α, β, γ isoforms), calculate the ratio of hyperphosphorylated (γ) to hypophosphorylated (α) forms as an additional measure of phosphorylation status. This integrated analytical approach provides comprehensive insight into EIF4EBP1 regulation under experimental conditions .

What statistical methods are most appropriate for analyzing EIF4EBP1 expression data in relation to clinical outcomes?

For analyzing EIF4EBP1 expression data in relation to clinical outcomes, employ a tiered statistical approach that matches analysis methods to specific research questions. For survival analysis, which has demonstrated significant associations between high EIF4EBP1 expression and poor outcomes in cancer patients, use Kaplan-Meier curves with log-rank tests to visualize and compare survival distributions between high and low expression groups. Determine expression thresholds objectively using methods such as median split or optimal cutpoint analysis (e.g., maximally selected rank statistics). For multivariable analysis, employ Cox proportional hazards regression to adjust for confounding clinical variables such as age, stage, and treatment history, reporting hazard ratios with confidence intervals. When analyzing EIF4EBP1 as a continuous variable, use Cox regression with restricted cubic splines to capture non-linear relationships with outcomes. For correlating EIF4EBP1 with other biomarkers or clinical parameters, use appropriate tests based on data distribution (Pearson or Spearman correlation for continuous variables, t-tests or Mann-Whitney for categorical comparisons). For predictive modeling, consider machine learning approaches such as random forests or support vector machines that can integrate EIF4EBP1 with other biomarkers. Validate findings through internal validation (bootstrapping, cross-validation) and, ideally, in independent external cohorts. This comprehensive statistical approach has successfully demonstrated EIF4EBP1's prognostic significance in multiple cancer types, particularly its association with tamoxifen resistance in breast cancer .