EIF4EBP1 Antibody, Biotin conjugated

What is EIF4EBP1 and why is it important in translation regulation?

EIF4EBP1 (4E-BP1) functions as a repressor of translation initiation by regulating eIF4E activity. In its hypophosphorylated state, EIF4EBP1 competes with EIF4G1/EIF4G3 and strongly binds to EIF4E, preventing the formation of the eIF4F complex and thereby repressing translation. When hyperphosphorylated, EIF4EBP1 dissociates from eIF4E, allowing interaction with EIF4G1/EIF4G3 and enabling translation initiation . This phosphorylation-dependent regulatory mechanism makes EIF4EBP1 a critical control point for protein synthesis in response to various stimuli, including hormones and growth factors that signal through MAP kinase and mTORC1 pathways . The protein serves as a rapamycin-sensitive downstream target of TOR (Target of rapamycin) and plays a fundamental role in cellular decisions regarding which mRNAs are translated under different physiological conditions .

What are the advantages of using biotin-conjugated antibodies for EIF4EBP1 detection?

Biotin-conjugated antibodies offer several significant advantages for EIF4EBP1 detection in research applications. This conjugation-ready format is specifically designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, making these antibodies highly versatile across multiple detection platforms . The strong interaction between biotin and streptavidin provides substantial signal amplification, enhancing detection sensitivity when working with proteins that may be expressed at low levels. For capture-based applications, biotin-labeled proteins can be efficiently isolated using streptavidin agarose resin . Additionally, biotin-conjugated antibodies excel in applications requiring downstream conjugation to detection systems and are particularly valuable for multiplex imaging applications, where simultaneous detection of multiple targets is essential . The stable nature of the biotin-streptavidin interaction also makes these conjugates more resilient to harsh washing conditions compared to direct conjugates.

What applications are biotin-conjugated EIF4EBP1 antibodies most suitable for?

Biotin-conjugated EIF4EBP1 antibodies demonstrate optimal performance in several specific applications. They are particularly well-suited for immunohistochemistry on paraffin-embedded tissues (IHC-P), as evidenced by successful staining protocols in formalin-fixed tissues . These antibodies also excel in sandwich ELISA (sELISA) applications, where they can be used for sensitive quantification of EIF4EBP1 with standard curves generating reliable background-subtracted data values . In protein-protein interaction studies, biotin-conjugated antibodies facilitate the capture of EIF4EBP1 and its binding partners using streptavidin-based affinity purification . The versatility of these conjugates extends to flow cytometry, where they can be used for intracellular staining to assess EIF4EBP1 levels at single-cell resolution. For researchers employing multiplex imaging techniques, the biotin conjugation provides a platform for sequential or simultaneous detection of EIF4EBP1 alongside other proteins of interest in complex tissue environments.

How does the phosphorylation state of EIF4EBP1 affect antibody binding?

The phosphorylation state of EIF4EBP1 can substantially impact antibody binding efficacy depending on the specific epitope recognized by the antibody. EIF4EBP1 undergoes phosphorylation following stimulation by mitogens such as insulin, which induces conformational changes in the protein structure . Antibodies targeting phosphorylation-dependent epitopes will only recognize EIF4EBP1 when the specific residue(s) are phosphorylated, making them valuable for studying activation states. Conversely, antibodies targeting regions that become masked or structurally altered upon phosphorylation may show diminished binding to hyperphosphorylated forms. When selecting antibodies for phosphorylation research, it is crucial to determine whether total EIF4EBP1 detection or phosphorylation-specific detection is needed. In Western blotting applications, especially when using techniques like Phos-Tag analysis, multiple bands may be observed representing different phosphorylation states of the protein . These migration patterns can provide valuable information about the relative abundance of differently phosphorylated EIF4EBP1 species in experimental samples.

What controls should be included when using EIF4EBP1 antibodies in experiments?

Rigorous experimental design with appropriate controls is essential when working with EIF4EBP1 antibodies. The following controls should be incorporated:

Additionally, when studying EIF4EBP1 in signaling contexts, samples treated with mTOR pathway activators (insulin, amino acids) or inhibitors (rapamycin, Torin1) provide functional validation of antibody specificity to phosphorylation changes.

How can biotin-conjugated EIF4EBP1 antibodies be optimized for multiplex imaging applications?

Optimizing biotin-conjugated EIF4EBP1 antibodies for multiplex imaging applications requires a systematic approach addressing several technical considerations:

First, thorough blocking of endogenous biotin is critical, particularly in biotin-rich tissues like liver, kidney, and brain. Commercial avidin/biotin blocking kits should be employed before antibody incubation to minimize background. Titration experiments must be conducted to determine the optimal antibody concentration that provides maximum specific signal while maintaining minimal background – typically starting from the 2.5-5.0 μg/ml range recommended for Western blotting applications .

For sequential multiplex protocols, complete inactivation of streptavidin-biotin interactions between detection rounds is essential. This can be achieved through heat-mediated reversal (90-100°C in glycine buffer, pH 2.5-3.0) or chemical methods (2-8M urea or 0.1-0.5M sodium hydroxide). When selecting detection reagents, streptavidin conjugates with spectrally distinct fluorophores should be chosen to minimize channel overlap and bleed-through.

Advanced signal amplification can be achieved through tiered approaches, such as biotin-streptavidin-biotin layering or tyramide signal amplification. The detection strategy should be customized based on EIF4EBP1 abundance, with low-expression contexts benefiting from more robust amplification systems. Finally, automated image analysis algorithms should be implemented to accurately quantify colocalization patterns and expression levels across different experimental conditions.

What are the technical considerations when using EIF4EBP1 antibodies to study the mTOR signaling pathway?

Studying the mTOR signaling pathway with EIF4EBP1 antibodies requires attention to several critical technical factors:

For comprehensive pathway analysis, multiple antibodies recognizing different phosphorylation sites should be employed. The standard sites include Thr37/46 (priming phosphorylation), followed by Thr70 and Ser65 (hierarchical phosphorylation). Phos-Tag gel electrophoresis provides superior separation of differentially phosphorylated forms, requiring extended run times (4 hours at 90V) for optimal band resolution .

Correlation with other mTOR targets (such as S6K1 phosphorylation) provides context for interpreting EIF4EBP1 results. Importantly, researchers should be aware that different mTOR inhibitors have varying effects on EIF4EBP1 phosphorylation – first-generation rapalogs (rapamycin, everolimus) incompletely inhibit EIF4EBP1 phosphorylation, while second-generation ATP-competitive inhibitors (Torin1, INK128) more effectively block all phosphorylation sites. This differential response makes EIF4EBP1 an excellent biomarker for distinguishing between partial and complete mTOR inhibition.

How do different fixation methods affect the performance of biotin-conjugated EIF4EBP1 antibodies in IHC?

Fixation methodology significantly impacts the performance of biotin-conjugated EIF4EBP1 antibodies in immunohistochemical applications. Formalin fixation, while preserving tissue morphology, creates protein cross-links that can mask epitopes, especially phosphorylation sites. For paraffin-embedded tissues, these antibodies have demonstrated compatibility with appropriate antigen retrieval methods . Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is typically effective for recovering EIF4EBP1 epitopes, though optimization for specific phospho-epitopes may require EDTA-based buffers (pH 9.0).

The timing between tissue collection and fixation is critical for preserving EIF4EBP1 phosphorylation states, as rapid dephosphorylation occurs post-mortem. For phosphorylation-specific studies, samples should be fixed within minutes of collection, ideally with phosphatase inhibitors included in the fixation protocol. Alternative fixatives like Bouin's solution or methanol-based fixatives may better preserve certain phospho-epitopes but can affect tissue morphology and increase non-specific binding.

Notably, endogenous biotin levels vary with fixation methods and can contribute to background signal. Paraformaldehyde fixation typically preserves more endogenous biotin than formalin, necessitating more rigorous blocking steps. The optimal fixation protocol should be empirically determined for each tissue type, balancing epitope preservation with structural integrity and minimizing background from endogenous biotin.

What are the challenges in detecting different phosphorylation states of EIF4EBP1 in clinical samples?

Detecting different phosphorylation states of EIF4EBP1 in clinical samples presents multiple technical challenges that researchers must address for reliable results:

Tissue heterogeneity within clinical samples adds complexity, as cells in different microenvironments may exhibit varying EIF4EBP1 phosphorylation patterns. Standard formalin fixation can mask phospho-epitopes, requiring optimized antigen retrieval protocols specific to each phosphorylation site. Additionally, the dynamic range of phosphorylation detection in IHC is limited compared to quantitative methods like Western blotting with Phos-Tag technology .

To overcome these challenges, researchers should employ multiple antibodies targeting different phosphorylation sites and correlate findings with downstream functional markers of translation activity. Digital image analysis with machine learning algorithms can help quantify subtle differences in staining patterns across heterogeneous tissues, providing more nuanced assessment of EIF4EBP1 activation states in clinical contexts.

How can researchers distinguish between EIF4EBP1 and other related 4E-binding proteins in complex biological samples?

Distinguishing between EIF4EBP1 and other related 4E-binding proteins (4EBP2 and 4EBP3) in complex biological samples requires strategic antibody selection and validation approaches:

Antibody specificity is paramount – researchers should select antibodies raised against regions with minimal sequence homology between family members. The C-terminal region of EIF4EBP1 offers good specificity as recognized by certain antibodies . Western blot analysis provides initial validation, with EIF4EBP1 typically appearing at approximately 20 kDa in mouse 3T3 cell lysates, despite its predicted molecular weight of 13 kDa . This migration discrepancy results from post-translational modifications and helps distinguish it from other family members.

For definitive validation, knockdown or knockout approaches provide the gold standard. siRNA or CRISPR-based depletion of EIF4EBP1 should eliminate the specific signal while leaving signals from other family members intact. Immunoprecipitation followed by mass spectrometry analysis can unambiguously identify the captured protein and detect any cross-reactivity with other 4E-binding proteins.

Expression pattern analysis can provide additional discrimination, as the three 4E-binding proteins show tissue-specific distribution patterns. Finally, functional validation through assays measuring specific binding to eIF4E can complement immunological detection methods, as the different family members exhibit varying binding affinities under different cellular conditions.

What is the optimal protocol for using biotin-conjugated EIF4EBP1 antibodies in Western blotting?

The optimal Western blotting protocol for biotin-conjugated EIF4EBP1 antibodies requires careful optimization of several parameters:

Sample Preparation:

• Include phosphatase inhibitors to preserve phosphorylation states

• Lyse cells directly in Laemmli buffer for immediate denaturation

• For phosphorylation analysis, prepare parallel samples with/without phosphatase treatment

Gel Electrophoresis:

• Use 12-15% polyacrylamide gels for optimal separation

• For phosphorylation analysis, employ Phos-Tag gels run at 90V for 4 hours

• Include positive controls such as insulin-stimulated cell lysates

Transfer and Blocking:

• Transfer to PVDF membranes (preferable over nitrocellulose for phospho-epitopes)

• Block with 5% BSA in TBST (superior to milk for phospho-specific detection)

• Include avidin/biotin blocking step to reduce endogenous biotin background

Antibody Incubation:

• Start with 2.5-5.0 μg/ml concentration as recommended for unconjugated antibodies

• Incubate overnight at 4°C in 5% BSA/TBST

• Wash extensively (4-5 times for 5 minutes each) with TBST

Detection:

• Use HRP-conjugated or fluorescently-labeled streptavidin at optimized dilution

• Expect bands at approximately 20 kDa, despite 13 kDa predicted weight

• Multiple bands may represent different phosphorylation states

This protocol should result in specific detection of EIF4EBP1 with minimal background. For quantitative analysis, include housekeeping protein controls and analyze using densitometry software.

How should samples be prepared for immunohistochemistry with biotin-conjugated EIF4EBP1 antibodies?

Sample preparation for immunohistochemistry with biotin-conjugated EIF4EBP1 antibodies requires meticulous attention to preservation of both tissue architecture and target epitopes:

Tissue Collection and Fixation:

• Minimize ischemic time to preserve phosphorylation states

• Fix in 10% neutral buffered formalin for 24-48 hours

• For phospho-specific detection, consider adding phosphatase inhibitors to fixative

Processing and Sectioning:

• Process tissues using standard paraffin embedding protocols

• Section at 4-5 μm thickness onto charged slides

• Store sections at room temperature or 4°C with desiccant

Pretreatment:

• Deparaffinize completely in xylene and rehydrate through graded alcohols

• Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Block endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes

Blocking:

• Block endogenous biotin using commercial avidin/biotin blocking kits (crucial step)

• Apply protein block (2-5% normal serum from secondary antibody species)

• For fatty tissues, include 0.1% Triton X-100 in blocking buffer

Antibody Incubation:

• Apply optimized concentration of biotin-conjugated EIF4EBP1 antibody

• Incubate overnight at 4°C in humidity chamber

• Include negative controls (isotype control or antibody diluent only)

These preparations have been shown to yield successful results in formalin-fixed paraffin-embedded normal human tissues , allowing precise localization of EIF4EBP1 within cellular compartments.

What strategies can be employed to reduce background when using biotin-conjugated antibodies?

Minimizing background when using biotin-conjugated antibodies requires a multi-faceted approach addressing several potential sources of non-specific signal:

Endogenous Biotin Blocking:

• Use commercial avidin/biotin blocking kits before antibody application

• For tissues with exceptionally high biotin content (liver, kidney, brain), consider extended blocking

• Streptavidin-only control slides help identify endogenous biotin contribution

Antibody Optimization:

• Titrate antibody concentration starting from 2.5-5.0 μg/ml recommended range

• Prepare parallel dilution series to identify optimal signal-to-noise ratio

• Consider overnight incubation at 4°C rather than shorter incubations at higher temperatures

Buffer Optimization:

• Include 0.1-0.3% Triton X-100 or 0.05-0.1% Tween-20 in washing buffers

• Use BSA instead of milk proteins when detecting phosphorylated epitopes

• Consider adding 5% normal serum from the species producing streptavidin conjugate

Additional Strategies:

• For tissues with high autofluorescence, treat with Sudan Black B before antibody application

• Use biotinylated secondary antibody with amplification rather than direct biotin conjugate for weak signals

• Implement stringent washing protocols (increased number and duration of washes)

• For problematic samples, consider alternative detection methods such as polymer-based systems

Careful application of these strategies can significantly improve signal-to-noise ratio when using biotin-conjugated EIF4EBP1 antibodies, enhancing the reliability and interpretability of experimental results.

How can researchers validate the specificity of their EIF4EBP1 antibody?

Comprehensive validation of EIF4EBP1 antibody specificity requires multiple complementary approaches:

Western Blot Analysis:

• Verify single band at expected molecular weight (~20 kDa in mouse 3T3 cell lysates)

• Compare migration pattern with recombinant EIF4EBP1 protein standard

• Assess cross-reactivity with related family members (4EBP2, 4EBP3)

Genetic Validation:

• Perform siRNA or shRNA knockdown of EIF4EBP1

• Generate CRISPR/Cas9 knockout cell lines as definitive negative controls

• Overexpress tagged EIF4EBP1 as positive control

Peptide Competition:

• Pre-incubate antibody with immunizing peptide (14 amino acids near human 4EBP1 C-terminus)

• Compare signal with and without peptide competition

• Include irrelevant peptide as control for non-specific blocking

Signaling Pathway Modulation:

• Treat cells with mTOR activators (insulin, amino acids) and inhibitors (rapamycin, Torin1)

• Verify expected changes in phosphorylation pattern

• Correlate with other markers of mTOR pathway activation

Multiple Antibody Comparison:

• Test multiple antibodies targeting different EIF4EBP1 epitopes

• Compare staining patterns across different applications

• Concordance between independent antibodies increases confidence in specificity

This multi-modal validation approach provides robust evidence for antibody specificity and helps researchers interpret experimental results with greater confidence.

What are the recommended approaches for quantifying EIF4EBP1 expression levels in different experimental settings?

Quantification of EIF4EBP1 expression requires tailored approaches for different experimental platforms:

Western Blotting:

• Perform densitometry analysis relative to housekeeping proteins

• Use standard curves with recombinant protein for absolute quantification

• For phosphorylation studies, calculate ratios between phosphorylated and total protein

• Consider Phos-Tag gels for separation and quantification of different phosphorylation states

Immunohistochemistry:

• Employ digital image analysis with standardized acquisition parameters

• Quantify using H-score method (percentage of positive cells × staining intensity)

• Use machine learning algorithms for automated scoring in heterogeneous tissues

• Include calibration standards on each slide for normalization between batches

ELISA-Based Methods:

• Develop standard curves using recombinant EIF4EBP1 protein

• Plot background-subtracted data values with appropriate statistical analysis

• Employ sandwich ELISA format for increased specificity

• Consider multiplex assays for simultaneous quantification of multiple phosphorylation sites

Flow Cytometry:

• Gate on relevant cell populations

• Quantify median fluorescence intensity relative to isotype controls

• Use phospho-flow techniques for single-cell resolution of phosphorylation states

• Consider fluorescence standardization beads for absolute quantification

RT-qPCR (mRNA level):

• Design primers spanning exon-exon junctions

• Normalize to validated reference genes

• Compare with protein levels to assess post-transcriptional regulation

• Particularly valuable for expression analysis in clinical samples

These quantification strategies should be selected based on the specific research question, required sensitivity, and available sample types.

How is EIF4EBP1 expression linked to prognosis in cancer research?

When focusing specifically on Group 3 and Group 4 medulloblastoma patients, high EIF4EBP1 expression maintained its association with poor outcome (p-value = 2.8e-03 and p-value = 7.9e-03) . Notably, this prognostic significance was particularly pronounced in Group 3 medulloblastoma patients (p-value = 0.025), while showing no significant association in Group 4, WNT, or SHH medulloblastoma subgroups . This pattern suggests context-dependent prognostic value, potentially reflecting differential roles of translation regulation in specific tumor molecular subtypes.

The prognostic significance of EIF4EBP1 likely relates to its fundamental role as a "repressor of translation initiation that regulates EIF4E activity" . Dysregulation of this critical node in translation control can promote the synthesis of proteins involved in survival, proliferation, and metastasis, driving more aggressive disease phenotypes. This relationship between EIF4EBP1 expression and clinical outcomes underscores the importance of translation regulation in cancer progression and identifies EIF4EBP1 as a potential biomarker for risk stratification.

What insights have EIF4EBP1 antibodies provided about translation regulation in stress conditions?

EIF4EBP1 antibodies have revealed crucial mechanisms of translation regulation during cellular stress responses. Under stress conditions, the hypophosphorylated form of EIF4EBP1 binds to eIF4E, inhibiting cap-dependent translation initiation . This regulatory mechanism allows cells to conserve energy and resources while prioritizing the synthesis of stress-response proteins, many of which utilize cap-independent translation mechanisms.

Studies employing phospho-specific EIF4EBP1 antibodies have demonstrated that diverse stressors, including nutrient deprivation, hypoxia, and endoplasmic reticulum (ER) stress, modulate EIF4EBP1 phosphorylation status. In the context of integrated stress response (ISR), GSK-3 inhibition has been shown to modulate protein translation regulation under ER stress conditions . While the direct interplay between GSK-3 and EIF4EBP1 was not explicitly described in the search results, both are implicated in the broader translation regulatory network responding to cellular stress.

The ability to track EIF4EBP1 phosphorylation status using specific antibodies has illuminated how various signaling pathways converge on translation control during stress adaptation. For example, the phosphorylation of EIF4EBP1 "following stimulation by mitogens such as insulin, results in the release of eIF-4E from 4EBP1, thereby enabling the translation complex to assemble" . This mechanistic insight helps explain how cells balance stress response with growth signals, maintaining translational homeostasis under varying physiological conditions.

How are EIF4EBP1 antibodies being used to study drug resistance mechanisms?

EIF4EBP1 antibodies have become instrumental tools for investigating drug resistance mechanisms, particularly in the context of targeted therapies affecting the mTOR signaling pathway. As EIF4EBP1 is "a rapamycin-sensitive downstream target of TOR" , antibodies recognizing its total and phosphorylated forms provide direct insight into mTOR inhibitor efficacy and the emergence of resistance.

In cancer treatment studies, phospho-specific EIF4EBP1 antibodies enable researchers to monitor whether cells maintain mTOR signaling despite the presence of inhibitory drugs. Persistent phosphorylation of EIF4EBP1 despite treatment often indicates the development of bypass mechanisms or mutations that render cancer cells resistant to therapy. Different classes of mTOR inhibitors show varying effects on EIF4EBP1 phosphorylation – first-generation rapalogs incompletely block EIF4EBP1 phosphorylation, while newer ATP-competitive inhibitors achieve more complete inhibition. This differential response makes EIF4EBP1 an excellent biomarker for distinguishing between drug classes and identifying specific resistance mechanisms.

The mechanistic understanding that "phosphorylation of 4EBP1 following stimulation by mitogens such as insulin, results in the release of eIF-4E from 4EBP1" has highlighted how alternative growth factor signaling pathways can potentially overcome drug-induced inhibition. Additionally, longitudinal monitoring of patient samples using EIF4EBP1 antibodies can track the evolution of resistance during treatment, guiding therapeutic decision-making and the development of more effective combination strategies.

What role does EIF4EBP1 play in neurological disease models?

Beyond cancer, EIF4EBP1 functions at the intersection of several signaling pathways implicated in neurological disorders. As a downstream effector of mTOR, which is crucial for synaptic plasticity and memory formation, EIF4EBP1 influences normal neuronal development and function. Dysregulation of this pathway is implicated in neurodevelopmental disorders characterized by altered protein synthesis rates.

The phosphorylation state of EIF4EBP1, detectable with phospho-specific antibodies, serves as a readout of translation regulation in neuronal models under various stress conditions. Research has demonstrated connections between integrated stress response (ISR) pathways and translation regulation under ER stress conditions , which is particularly relevant for neurodegenerative diseases where protein folding stress is a common pathogenic mechanism. By regulating which mRNAs are translated under different conditions, EIF4EBP1 helps determine neuronal fate decisions between adaptation, synaptic remodeling, and apoptosis.

How can EIF4EBP1 antibodies be used to evaluate mTOR inhibitor efficacy?

EIF4EBP1 antibodies provide valuable tools for evaluating mTOR inhibitor efficacy across diverse experimental and clinical applications:

Mechanism-Based Assessment:
EIF4EBP1 serves as "a rapamycin-sensitive downstream target of TOR" , making it an ideal biomarker for monitoring mTOR inhibition. Phospho-specific antibodies targeting multiple sites (Thr37/46, Ser65, Thr70) can assess the degree and pattern of inhibition following drug treatment. Unlike some mTOR substrates, EIF4EBP1 phosphorylation is more resistant to first-generation rapalogs but sensitive to second-generation ATP-competitive inhibitors, making it valuable for distinguishing between drug classes.

Technical Approaches:
When combined with Phos-Tag gel electrophoresis technology, which allows "optimal separation of bands" , EIF4EBP1 antibodies can reveal the distribution of differently phosphorylated species, providing a detailed view of partial versus complete inhibition. This technique enables visualization of multiple phosphorylation states simultaneously, offering more comprehensive assessment than site-specific phospho-antibodies alone.

Translational Applications:
In patient-derived samples, immunohistochemistry with EIF4EBP1 antibodies can evaluate drug penetration and target engagement in heterogeneous tissues. Additionally, combining EIF4EBP1 phosphorylation assessment with functional readouts of cap-dependent translation (such as analysis of known eIF4E-dependent transcripts) provides a more complete picture of the biological consequences of mTOR inhibition.

Resistance Monitoring: Serial sampling with EIF4EBP1 antibody analysis can track the emergence of resistance mechanisms during treatment, potentially identifying patients who might benefit from alternative strategies or combination approaches before clinical progression becomes evident.