EIF4EBP2 Antibody

What is EIF4EBP2 and what is its function in cellular processes?

EIF4EBP2 (Eukaryotic translation initiation factor 4E-binding protein 2), also known as 4E-BP2, functions as a repressor of translation initiation involved in synaptic plasticity, learning, and memory formation . It regulates EIF4E activity by preventing its assembly into the eIF4F complex through a competitive binding mechanism with EIF4G1/EIF4G3 . The hypophosphorylated form of EIF4EBP2 strongly binds to EIF4E to repress translation, while the hyperphosphorylated form dissociates from EIF4E, allowing interaction with EIF4G1/EIF4G3 and initiating translation . Notably, EIF4EBP2 is enriched in brain tissue where it acts as a critical regulator of synapse activity and neuronal stem cell renewal through its translation repression capabilities .

What molecular weight should I expect to observe for EIF4EBP2 in Western blots?

When performing Western blot analysis, researchers should expect to observe EIF4EBP2 at a molecular weight range of 15-20 kDa . While the calculated molecular weight based on amino acid sequence is approximately 13 kDa (from its 120 amino acids) , post-translational modifications—particularly phosphorylation states—can result in the observed higher molecular weight bands. This discrepancy between calculated and observed molecular weight is important to consider when interpreting Western blot results, as it may reflect different phosphorylation states of the protein rather than non-specific binding or degradation products.

What species reactivity can I expect from commonly available EIF4EBP2 antibodies?

Available EIF4EBP2 antibodies demonstrate various cross-reactivity profiles across species. Cell Signaling's antibody #2845 shows reactivity with human, mouse, rat, monkey, and bovine samples . Proteintech's 15628-1-AP has been validated specifically for human, mouse, and rat samples . Some antibodies from other manufacturers offer broader species cross-reactivity, with certain products validated for up to ten species including human, mouse, cow, dog, pig, rat, goat, rabbit, zebrafish, and monkey . When selecting an antibody for your experimental system, it is critical to choose one with validated reactivity for your species of interest to ensure experimental success and data reliability.

What are the validated applications for EIF4EBP2 antibodies?

EIF4EBP2 antibodies have been validated for multiple experimental applications, with specific dilution recommendations varying by manufacturer. Western blotting (WB) is the most commonly validated application, with recommended dilutions typically ranging from 1:1000 to 1:4000 . Immunoprecipitation (IP) is supported by some antibodies, such as CST's #2845, with a recommended dilution of 1:100 . Immunohistochemistry on paraffin-embedded samples (IHC-P) is another validated application, with dilutions ranging from 1:200 to 1:800 depending on the antibody and tissue type . Some antibodies are also validated for ELISA applications . Researchers should carefully review the validation data provided by manufacturers for their specific experimental conditions, as performance may vary based on sample type and preparation method.

How should I optimize Western blot protocols for EIF4EBP2 detection?

For optimal Western blot detection of EIF4EBP2, consider the following methodology: Begin with sample preparation using a buffer containing phosphatase inhibitors, particularly if examining phosphorylation states. Use 10-15% SDS-PAGE gels to achieve good separation in the 15-20 kDa range where EIF4EBP2 is detected . Transfer proteins to a PVDF or nitrocellulose membrane at lower voltage for better transfer of small proteins. For primary antibody incubation, a dilution of 1:1000-1:4000 is typically recommended for most EIF4EBP2 antibodies, with overnight incubation at 4°C providing optimal results . Include positive control samples such as HepG2 cell lysate, which has been validated to express detectable levels of EIF4EBP2 . When interpreting results, be aware that multiple bands may represent different phosphorylation states rather than non-specific binding.

What are the key considerations for successful immunohistochemistry with EIF4EBP2 antibodies?

For successful immunohistochemistry (IHC) using EIF4EBP2 antibodies, several methodological factors should be considered. First, ensure proper tissue fixation—typically 10% neutral buffered formalin is recommended, though optimization may be required for brain tissues where EIF4EBP2 is highly expressed . Antigen retrieval is critical; heat-induced epitope retrieval in citrate buffer (pH 6.0) is often effective for EIF4EBP2 detection. For primary antibody incubation, dilutions of 1:200-1:800 are typically recommended , with overnight incubation at 4°C often yielding better results than shorter incubations. Include appropriate positive control tissues (brain sections are ideal) and negative controls (primary antibody omission and isotype controls). For detection systems, both DAB chromogenic and fluorescent secondary antibodies have been successfully used with EIF4EBP2 antibodies, though signal amplification may be necessary for detecting lower expression levels.

Why might I observe multiple bands or band shifts in Western blots using EIF4EBP2 antibodies?

Multiple bands or band shifts observed in Western blots with EIF4EBP2 antibodies frequently represent different phosphorylation states rather than non-specific binding . EIF4EBP2 is regulated through hierarchical phosphorylation at multiple sites, similar to 4E-BP1, where phosphorylation by FRAP/mTOR at specific threonine residues primes the protein for subsequent phosphorylation at other sites . These various phosphorylation states cause mobility shifts during electrophoresis, resulting in bands appearing between 15-20 kDa . When studying signaling pathways affecting EIF4EBP2 phosphorylation, these multiple bands provide valuable information about activation states. To confirm band specificity, researchers can treat samples with phosphatase, use phospho-specific antibodies as complementary tools, or include positive control samples with known phosphorylation states. These approaches help distinguish between specific EIF4EBP2 signals and potential cross-reactivity with related family members like 4E-BP1 or 4E-BP3.

How can I minimize background and improve signal specificity in immunohistochemistry?

To minimize background and improve signal specificity in immunohistochemistry with EIF4EBP2 antibodies, implement these methodological refinements: Begin with thorough blocking using 5-10% normal serum from the same species as the secondary antibody, supplemented with 0.1-0.3% Triton X-100 for improved penetration in brain tissues where EIF4EBP2 is enriched . Optimize antibody concentration through careful titration; while manufacturer recommendations suggest dilutions of 1:200-1:800 , preliminary testing with smaller dilution increments can identify the optimal concentration for your specific tissue and fixation conditions. Include additional blocking steps with avidin/biotin if using biotinylated detection systems. Extend washing steps (at least three 10-minute washes) between antibody incubations to reduce non-specific binding. Consider using more specific detection methods such as tyramide signal amplification, which can allow for lower primary antibody concentrations while maintaining detection sensitivity. Finally, always include appropriate negative controls by omitting the primary antibody or using isotype-matched control antibodies to distinguish true signal from background.

How should I store and handle EIF4EBP2 antibodies to maintain their performance?

Proper storage and handling of EIF4EBP2 antibodies is crucial for maintaining their performance over time. Most EIF4EBP2 antibodies should be stored at -20°C according to manufacturer recommendations . The storage buffer typically contains PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain antibody stability . Antibodies are generally stable for one year after shipment when stored properly. For the 20μl size antibodies, manufacturers often include 0.1% BSA in the formulation to enhance stability . Avoid repeated freeze-thaw cycles by aliquoting the antibody upon first use, although some formulations specify that aliquoting is unnecessary for -20°C storage . When handling the antibody for experiments, always keep it on ice and return to storage promptly. For diluted working solutions, prepare fresh on the day of experiment or store at 4°C for no more than 1-2 weeks. Following these storage guidelines will help preserve antibody activity and ensure consistent experimental results.

How can I use EIF4EBP2 antibodies to study translation regulation in neuronal function?

EIF4EBP2 antibodies provide powerful tools for investigating translation regulation in neuronal function, particularly since EIF4EBP2 is enriched in brain tissue and plays critical roles in synaptic plasticity, learning, and memory formation . A comprehensive experimental approach would combine multiple techniques. For immunohistochemistry in brain sections, use dilutions between 1:200-1:800 to visualize regional expression patterns and co-localize with synaptic markers . In cultured neurons, combine immunocytochemistry with proximity ligation assays to visualize EIF4EBP2-EIF4E interactions at specific subcellular compartments like dendritic spines. For biochemical analysis, perform polysome profiling of neuronal lysates followed by Western blotting (1:1000-1:4000 dilution) to assess EIF4EBP2 distribution between actively translating and non-translating fractions . Use phospho-specific antibodies to monitor changes in EIF4EBP2 phosphorylation state in response to neuronal activity, learning paradigms, or pharmacological manipulations of signaling pathways. Additionally, combine immunoprecipitation (1:100 dilution) with RNA sequencing to identify transcripts whose translation is regulated by EIF4EBP2 in neuronal contexts . This multi-faceted approach enables researchers to comprehensively characterize how EIF4EBP2-mediated translation regulation contributes to neuronal function and synaptic plasticity.

How can I differentiate between the functions of EIF4EBP1, EIF4EBP2, and EIF4EBP3 in my research?

Differentiating between EIF4EBP family members requires careful antibody selection and experimental design. First, select antibodies with validated specificity for each family member, particularly for EIF4EBP2, which shares high sequence homology with EIF4EBP1 and EIF4EBP3 . Verify antibody specificity through Western blotting in tissues with differential expression profiles—while EIF4EBP2 is enriched in brain tissue, other family members may show different tissue distributions . For functional studies, design siRNA or CRISPR knockout experiments targeting each family member individually, then validate knockdown specificity using your validated antibodies. When examining phosphorylation, note that while phosphorylation sites may be conserved across family members, the regulation and kinetics of phosphorylation can differ . EIF4EBP2 phosphorylation is regulated similarly to EIF4EBP1, but with distinct patterns in neuronal contexts . Use immunoprecipitation followed by mass spectrometry to identify unique binding partners for each family member. Finally, tissue-specific analysis is crucial—EIF4EBP2's predominant role in brain function can be distinguished from other family members by examining neuronal translation regulation, synaptic plasticity, and memory formation contexts where it plays a specialized role .

What approaches can I use to study the role of EIF4EBP2 in the mTOR signaling pathway?

To study EIF4EBP2's role in mTOR signaling, implement these methodological approaches: Begin with Western blot analysis (1:1000-1:4000 dilution) to monitor EIF4EBP2 phosphorylation states in response to mTOR pathway modulators, such as rapamycin (an mTORC1 inhibitor) or growth factors . Phosphorylation at conserved sites (similar to those in 4E-BP1) can be detected as mobility shifts between 15-20 kDa . Perform co-immunoprecipitation (1:100 dilution) to assess EIF4EBP2 interaction with EIF4E under various mTOR activation states . For dynamic studies, use kinase assays with recombinant mTOR and EIF4EBP2 to characterize phosphorylation kinetics. Implement proximity ligation assays to visualize EIF4EBP2-EIF4E interactions in situ following mTOR pathway manipulation. For functional analysis, combine polysome profiling with RNA sequencing to identify transcripts whose translation is specifically regulated by EIF4EBP2 in an mTOR-dependent manner. In neuronal systems, where EIF4EBP2 is enriched, examine how mTOR-dependent phosphorylation of EIF4EBP2 affects synaptic protein synthesis, spine morphology, and plasticity . Use phospho-mimetic and phospho-deficient EIF4EBP2 mutants to distinguish between mTOR-dependent and independent functions. These approaches provide complementary insights into EIF4EBP2's specific role in mediating translational control downstream of mTOR signaling.

What positive and negative controls should I include when working with EIF4EBP2 antibodies?

For rigorous experimental design with EIF4EBP2 antibodies, include these essential controls: For positive controls in Western blotting, use HepG2 cell lysates, which have been validated to express detectable levels of endogenous EIF4EBP2 . Brain tissue lysates are also excellent positive controls given EIF4EBP2's enrichment in neural tissues . For immunohistochemistry positive controls, hippocampal and cortical brain sections provide reliable EIF4EBP2 expression . For negative controls, include samples treated with EIF4EBP2-specific siRNA or from knockout models when available. Technical negative controls should include primary antibody omission and isotype-matched irrelevant antibodies to assess non-specific binding. For phosphorylation studies, include samples treated with phosphatase inhibitors (positive control) and phosphatase enzymes (negative control) to validate phospho-specific detection. When studying mTOR pathway regulation, include samples treated with mTOR inhibitors (like rapamycin) and activators (like insulin) to demonstrate expected phosphorylation changes. Additionally, recombinant EIF4EBP2 protein can serve as a standard for antibody validation, especially when testing new lots. Incorporating these comprehensive controls ensures reliable interpretation of EIF4EBP2 detection across experimental conditions.

How can I design experiments to study EIF4EBP2 phosphorylation states?

Designing experiments to study EIF4EBP2 phosphorylation states requires careful consideration of multiple factors. Begin by selecting appropriate lysis conditions that preserve phosphorylation status—use buffers containing phosphatase inhibitors such as sodium fluoride, sodium orthovanadate, and β-glycerophosphate. For Western blot analysis, use Phos-tag™ or similar phosphate-binding acrylamide gels to enhance separation of different phosphorylation states beyond the standard 15-20 kDa range observed with conventional SDS-PAGE . To manipulate phosphorylation states, treat cells with mTOR pathway modulators (rapamycin as inhibitor, insulin or serum as activators) prior to lysis . For time-course experiments, include multiple time points after stimulation to capture the dynamic nature of EIF4EBP2 phosphorylation. When available, use phospho-specific antibodies that recognize specific phosphorylation sites, although these might be more commonly available for EIF4EBP1 than EIF4EBP2 . To confirm band identity, include lambda phosphatase treatment controls. For functional correlation, pair phosphorylation analysis with cap-binding assays to determine how phosphorylation states affect EIF4EBP2 interaction with EIF4E. Finally, consider mass spectrometry approaches to identify all phosphorylation sites simultaneously, which can reveal previously uncharacterized modifications beyond those commonly studied.

What methodological approaches are best for studying EIF4EBP2 interactions with binding partners?

To study EIF4EBP2 interactions with binding partners, several complementary methodological approaches are recommended. Co-immunoprecipitation (co-IP) using EIF4EBP2 antibodies at 1:100 dilution is effective for pulling down protein complexes containing EIF4EBP2 and its binding partners like EIF4E . For the reverse approach, perform m7GTP cap pulldown assays to capture EIF4E and associated proteins, then probe for EIF4EBP2 using Western blotting (1:1000-1:4000 dilution) . For visualization of interactions in situ, implement proximity ligation assays (PLA) or fluorescence resonance energy transfer (FRET) in fixed cells or tissues, which are particularly valuable for examining interactions in specific subcellular compartments of neurons where EIF4EBP2 is enriched . For quantitative analysis of binding affinities, use surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) with recombinant proteins. To examine how phosphorylation affects interactions, compare binding profiles under various conditions—such as mTOR inhibition or activation—that alter EIF4EBP2 phosphorylation states . For comprehensive interactome analysis, combine immunoprecipitation with mass spectrometry to identify novel binding partners beyond the canonical translation factors. These approaches provide complementary insights into EIF4EBP2's biological role in translation regulation and potential moonlighting functions in other cellular processes.

What are the key differences between antibodies targeting different epitopes of EIF4EBP2?

Antibodies targeting different epitopes of EIF4EBP2 demonstrate important functional differences that should inform selection for specific research applications. N-terminal targeting antibodies (such as those recognizing AA 1-120 or AA 7-56) are valuable for detecting total EIF4EBP2 protein levels regardless of phosphorylation state . These antibodies typically recognize EIF4EBP2 in both its bound (to EIF4E) and unbound states, making them ideal for total protein expression studies. In contrast, antibodies targeting the C-terminal region (such as those recognizing AA 87-115) may have differential access to epitopes depending on protein conformation and binding status . Mid-region epitope antibodies (like those targeting AA 50 to C-terminus or AA 61-120) often provide a balance between detection efficiency and specificity . When studying phosphorylation, epitope choice becomes crucial—antibodies whose epitopes include or are adjacent to phosphorylation sites may show reduced binding when the protein is phosphorylated. The chosen epitope also impacts species cross-reactivity; antibodies targeting highly conserved regions demonstrate broader species reactivity, while those targeting variable regions provide greater specificity for particular species . These considerations highlight the importance of selecting antibodies with epitopes appropriate for your specific research question, whether focusing on total protein detection, phosphorylation status, or species-specific analysis.

Which EIF4EBP2 antibody characteristics are most important for different experimental applications?

The optimal EIF4EBP2 antibody characteristics vary significantly by experimental application, requiring careful selection based on specific research needs. For Western blotting, prioritize antibodies with high sensitivity in the 15-20 kDa range, such as Proteintech's 15628-1-AP (1:1000-1:4000 dilution) or Cell Signaling's #2845 (1:1000 dilution) . Antibody specificity is particularly critical given EIF4EBP2's homology with other family members, so validated antibodies with minimal cross-reactivity are essential. For immunoprecipitation studies, choose antibodies specifically validated for IP applications, such as Cell Signaling's #2845 at 1:100 dilution, which can effectively capture EIF4EBP2 protein complexes . For immunohistochemistry in neuronal tissues where EIF4EBP2 is enriched, select antibodies validated for IHC-P with recommended dilutions between 1:200-1:800 . Consider species reactivity based on your experimental model—while some antibodies show limited reactivity with human and mouse tissues only, others demonstrate broader cross-reactivity across multiple species . For phosphorylation studies, epitope location becomes critical; avoid antibodies whose epitopes include known phosphorylation sites if you aim to detect all forms of the protein. Finally, for multiplexing experiments, consider host species compatibility with other primary antibodies in your panel to avoid secondary antibody cross-reactivity issues.