EIF4ENIF1 Antibody

What is EIF4ENIF1 and what is its biological significance in research?

EIF4ENIF1 (Eukaryotic Translation Initiation Factor 4E Nuclear Import Factor 1), also known as 4E-T or Clast4, functions as a nucleocytoplasmic shuttle protein for the translation initiation factor eIF4E. This protein plays a critical role in mediating the nuclear import of eIF4E through interaction with the importin alpha-beta complex. EIF4ENIF1 is predominantly cytoplasmic, with its nuclear import regulated by a nuclear localization signal and nuclear export signals .

The protein has significant research importance due to its involvement in translation regulation, mRNA processing, and developmental biology. EIF4ENIF1 represses translation by binding to eIF4E and targets eIF4E to P bodies (cytoplasmic structures containing factors that degrade eIF4E-bound mRNA). Research has shown that depleting EIF4ENIF1 from cells results in increased mRNA stability, suggesting its role in regulating mRNA degradation pathways .

Research applications focus on its expression in various tissues, particularly in reproductive biology, where mutations in the EIF4ENIF1 gene have been associated with primary ovarian insufficiency in women .

Which antibody formats are available for EIF4ENIF1 research and what are their comparative advantages?

Current research involving EIF4ENIF1 utilizes several antibody formats, each with distinct advantages:

Researchers should select antibodies based on their specific application requirements. Rabbit polyclonal antibodies offer broader epitope recognition, potentially increasing detection sensitivity, while mouse antibodies may provide advantages in multi-color immunofluorescence experiments where host species differentiation is required .

What are the recommended storage and handling protocols to maintain EIF4ENIF1 antibody integrity?

To ensure optimal performance and longevity of EIF4ENIF1 antibodies, researchers should adhere to the following evidence-based storage and handling protocols:

• Storage temperature: Store antibodies at -20°C for long-term preservation. This temperature minimizes degradation while maintaining antibody function .

• Buffer composition: EIF4ENIF1 antibodies are typically supplied in phosphate buffered solution (pH 7.4) containing stabilizers (0.05%) and 50% glycerol to prevent freeze-thaw damage .

• Aliquoting: Divide antibody solutions into small working aliquots to avoid repeated freeze-thaw cycles, which can significantly reduce antibody activity .

• Shipping and receipt handling: Upon receiving shipped antibodies (typically with ice packs), immediately transfer to recommended storage conditions to prevent degradation .

• Shelf-life considerations: Most EIF4ENIF1 antibodies maintain validity for approximately 12 months when stored properly under recommended conditions .

These protocols ensure that the structural integrity and functional properties of the antibodies are preserved throughout experimental workflows .

What experimental conditions optimize Western blot detection of EIF4ENIF1?

Optimizing Western blot detection of EIF4ENIF1 requires attention to several critical parameters:

• Sample Preparation and Loading:

  • Validated cell lines include Jurkat, HeLa, HT29, and L-O2, which demonstrate reliable EIF4ENIF1 expression .

  • Nuclear extracts (particularly from HeLa S3) provide strong signal detection for nuclear-localized EIF4ENIF1 .

• Dilution Optimization:

  • For rabbit polyclonal antibodies: Use dilutions in the range of 1:500-1:2000 for optimal signal-to-noise ratio .

  • For mouse polyclonal antibodies: Follow manufacturer-specific recommendations for MaxPab antibodies .

• Molecular Weight Considerations:

  • The calculated molecular weight for EIF4ENIF1 is 88 kDa/108 kDa.

  • Importantly, the observed molecular weight is frequently 178 kDa, which differs significantly from the expected size.

  • Researchers should note that this discrepancy is normal, as the mobility of EIF4ENIF1 is affected by post-translational modifications .

  • Transfected lysates typically show bands around 108.35 kDa .

• Controls:

  • Include non-transfected lysates as negative controls when using transfection experiments .

  • Positive control using transfected 293T cell line expressing EIF4ENIF1 is recommended .

This methodological approach accounts for the unique characteristics of EIF4ENIF1 and addresses the common challenge of molecular weight discrepancies seen in Western blot detection .

How can researchers effectively use EIF4ENIF1 antibodies in immunofluorescence studies?

For optimal immunofluorescence detection of EIF4ENIF1, researchers should implement the following validated protocol:

• Cell Line Selection:

  • HeLa cells have been validated as reliable models for EIF4ENIF1 immunofluorescence studies .

  • These cells effectively demonstrate the dual cytoplasmic and nuclear localization of EIF4ENIF1 .

• Antibody Preparation:

  • Use purified antibody preparations to minimize background signal.

  • The recommended antibody concentration is 10 μg/ml for clear visualization of EIF4ENIF1 .

• Detection Strategy:

  • Apply standard immunofluorescence protocols with consideration for EIF4ENIF1's subcellular distribution.

  • Document both nuclear and cytoplasmic signals separately, as the protein shuttles between these compartments .

• Co-localization Studies:

  • Consider dual labeling with P-body markers to visualize EIF4ENIF1's role in mRNA degradation.

  • For nuclear studies, co-staining with eIF4E can demonstrate the protein's role in nuclear import .

• Image Analysis:

  • Quantify the nuclear-to-cytoplasmic ratio to assess shuttling dynamics.

  • Perform z-stack imaging to fully capture the three-dimensional distribution of the protein.

This approach enables detailed visualization of EIF4ENIF1's subcellular localization patterns, facilitating studies of its nucleocytoplasmic shuttling function and interactions with other cellular components .

How should researchers troubleshoot unexpected molecular weight variations when detecting EIF4ENIF1?

When encountering molecular weight discrepancies in EIF4ENIF1 detection, researchers should systematically address these variations through the following evidence-based approach:

• Understanding Normal Variation:

  • The calculated molecular weight of EIF4ENIF1 is 88-108 kDa, but the observed weight in Western blot applications is frequently 178 kDa .

  • This discrepancy is well-documented and considered normal for this protein.

• Mechanistic Explanation:

  • Post-translational modifications significantly affect protein mobility during electrophoresis.

  • EIF4ENIF1 may exist in multiple modified forms simultaneously, resulting in multiple bands on detection membranes .

• Validation Strategies:

  • Compare results with positive controls using transfected lysates expressing EIF4ENIF1 (expected at ~108.35 kDa) .

  • Include known EIF4ENIF1-expressing cell lines like Jurkat or HeLa as internal standards .

• Technical Adjustments:

  • Optimize gel percentage to better resolve the protein's actual molecular weight.

  • Consider using gradient gels (4-15%) to improve separation of high molecular weight proteins.

  • Modify running conditions (voltage/time) to achieve better resolution.

• Confirmatory Approaches:

  • If available, use multiple antibodies targeting different epitopes of EIF4ENIF1.

  • Consider complementary techniques like immunoprecipitation followed by mass spectrometry to confirm protein identity.

These approaches help researchers differentiate between genuine EIF4ENIF1 detection and potential artifacts, ensuring reliable experimental outcomes despite the characteristic molecular weight variations of this protein .

How can EIF4ENIF1 antibodies be utilized to investigate mRNA translation regulation mechanisms?

EIF4ENIF1 antibodies serve as powerful tools for dissecting the protein's role in translation regulation through several advanced methodological approaches:

• RNA Immunoprecipitation (RIP) Assays:

  • Utilize EIF4ENIF1 antibodies to immunoprecipitate the protein along with its bound mRNAs.

  • This allows identification of specific transcripts regulated by EIF4ENIF1.

  • Protocol adjustment: Since EIF4ENIF1 interacts with eIF4E and targets it to P-bodies for mRNA degradation, crosslinking steps should be optimized to capture these transient interactions .

• P-body Dynamics Studies:

  • EIF4ENIF1 antibodies can track the protein's localization to P-bodies, cytoplasmic structures involved in mRNA degradation.

  • Methodology involves immunofluorescence co-localization with known P-body markers.

  • This approach reveals how EIF4ENIF1 contributes to mRNA stability regulation, as depletion of EIF4ENIF1 has been shown to increase mRNA stability .

• Translation Repression Assays:

  • Employ EIF4ENIF1 antibodies in conjunction with polysome profiling to assess how EIF4ENIF1 affects the translation status of specific mRNAs.

  • This method can demonstrate EIF4ENIF1's repressive effect on translation through its binding to eIF4E .

• Mechanistic Studies of mRNA Decay:

  • Use EIF4ENIF1 antibodies in pulse-chase experiments tracking labeled mRNAs to quantify decay rates in the presence or absence of functional EIF4ENIF1.

  • This approach provides direct evidence of EIF4ENIF1's role in mRNA degradation pathways .

These methodologies leverage EIF4ENIF1 antibodies to illuminate the protein's multifaceted roles in post-transcriptional gene regulation, particularly its functions in translation repression and mRNA stability control .

What experimental approaches can elucidate EIF4ENIF1's nucleocytoplasmic shuttling function?

Investigating EIF4ENIF1's nucleocytoplasmic shuttling requires sophisticated experimental designs that track protein movement between cellular compartments:

• Live Cell Imaging with Fluorescent Fusion Proteins:

  • Complementary approach: Use antibodies to validate the localization patterns of endogenous EIF4ENIF1 compared to tagged versions.

  • Antibody-based immunofluorescence provides crucial baseline data for shuttling dynamics in unmodified cells.

• Subcellular Fractionation and Western Blotting:

  • Separate nuclear and cytoplasmic fractions using established protocols.

  • Apply EIF4ENIF1 antibodies in Western blotting to quantify the relative distribution between compartments.

  • This method provides quantitative assessment of EIF4ENIF1's localization under different cellular conditions .

• Immunofluorescence Studies of Nuclear Transport Signals:

  • EIF4ENIF1 contains one nuclear localization signal and two nuclear export signals.

  • Use site-directed mutagenesis to create signal variants, then employ antibodies to detect localization changes.

  • This approach can validate the functionality of these signals as demonstrated in previous research showing that truncating EIF4ENIF1 before the nuclear export signals results in nuclear retention .

• Co-immunoprecipitation with Transport Machinery:

  • Use EIF4ENIF1 antibodies to co-immunoprecipitate the protein with components of nuclear transport machinery.

  • This reveals direct interactions with importin alpha-beta complex that mediates nuclear import .

• Quantitative Shuttling Assays:

  • Apply techniques like fluorescence loss in photobleaching (FLIP) or fluorescence recovery after photobleaching (FRAP).

  • Use antibodies to validate shuttling rates of endogenous protein compared to fluorescently tagged versions.

These approaches collectively provide a comprehensive analysis of EIF4ENIF1's dynamic movement between nuclear and cytoplasmic compartments, which is critical to its function as a regulator of eIF4E localization and activity .

How can researchers investigate the role of EIF4ENIF1 in reproductive biology and ovarian function?

Research into EIF4ENIF1's role in reproductive biology, particularly ovarian function, can be approached through several sophisticated methodologies:

• Genetic Association Studies in Primary Ovarian Insufficiency (POI):

  • EIF4ENIF1 mutations, particularly nonsense mutations, have been associated with autosomal dominant POI .

  • Methodological approach: Use targeted sequencing of EIF4ENIF1 in POI patient cohorts, followed by functional validation of identified variants using antibody-based techniques.

  • Previous research identified a family with a nonsense mutation in EIF4ENIF1 that segregates with early menopause (ages 29-35) .

• Transcript and Protein Expression Analysis in Ovarian Tissue:

  • EIF4ENIF1 is expressed in human ovary tissue .

  • Methodology: Apply immunohistochemistry with validated EIF4ENIF1 antibodies to localize the protein within specific ovarian cell types and developmental stages.

  • Compare expression patterns between normal and pathological ovarian tissues.

• Functional Studies of Translation Regulation in Oocytes:

  • Based on homologues in model organisms (Drosophila Cup and mouse Clast4), EIF4ENIF1 likely plays a critical role in oocyte development .

  • Experimental approach: Use immunoprecipitation with EIF4ENIF1 antibodies to identify mRNAs specifically regulated in oocytes.

  • Analyze translation efficiency of these transcripts in normal versus EIF4ENIF1-depleted conditions.

• Mechanistic Investigation of Haploinsufficiency Effects:

  • Research suggests two potential mechanisms for ovarian insufficiency in EIF4ENIF1 mutations: haploinsufficiency or production of truncated protein .

  • Methodology: Use antibodies specific to N-terminal regions to detect truncated proteins in cellular models of EIF4ENIF1 mutations.

  • Assess downstream effects on mRNA stability, as EIF4ENIF1 depletion has been shown to increase mRNA stability .

• Comparative Studies with Model Organism Homologues:

  • The Drosophila Cup protein and mouse Clast4 protein are highly homologous to EIF4ENIF1 .

  • Experimental design: Use cross-reactive antibodies to perform comparative studies of protein function across species.

  • This approach leverages findings that Cup mutations in Drosophila disrupt oocyte growth and meiotic chromosome segregation .

These research approaches combine genetic, molecular, and cellular techniques to elucidate EIF4ENIF1's critical role in ovarian development and function, potentially advancing understanding of female reproductive disorders .

What validation methods should researchers employ to ensure EIF4ENIF1 antibody specificity?

To ensure experimental rigor when working with EIF4ENIF1 antibodies, researchers should implement this comprehensive validation strategy:

• Positive Control Validation:

  • Test antibodies on cell lines with confirmed EIF4ENIF1 expression, including:

    • U-251MG, HeLa, HT29, and L-O2 cells for rabbit polyclonal antibodies

    • Jurkat and HeLa cells for mouse polyclonal antibodies

  • Include EIF4ENIF1-transfected cell lines as strong positive controls (e.g., transfected 293T cells)

• Negative Control Testing:

  • Compare against non-transfected lysates when working with transfection experiments

  • Include isotype controls to identify non-specific binding

  • Consider siRNA knockdown of EIF4ENIF1 as a functional negative control

• Multi-technique Concordance:

  • Verify consistent results across multiple applications:

    • Western blot (WB)

    • Immunocytochemistry/Immunofluorescence (ICC-IF)

    • Immunohistochemistry (IHC)

  • Confirm that localization patterns match expected cytoplasmic and nuclear distribution

• Molecular Weight Verification:

  • Acknowledge the discrepancy between calculated (88-108 kDa) and observed (178 kDa) molecular weights

  • Verify that observed bands align with those reported in literature

  • Consider additional validation for unexpected banding patterns

• Cross-Reactivity Assessment:

  • Test antibodies on samples from different species when cross-reactivity is claimed (e.g., human and mouse)

  • Verify species-specific recognition patterns

This systematic validation approach ensures that experimental outcomes reflect true EIF4ENIF1 biology rather than artifacts of non-specific antibody binding .

How should researchers interpret contradictory results between different EIF4ENIF1 antibodies?

When faced with discrepancies between different EIF4ENIF1 antibodies, researchers should employ this systematic analysis framework:

• Epitope Mapping Analysis:

  • Determine the specific regions of EIF4ENIF1 targeted by each antibody.

  • Rabbit polyclonal antibodies may recognize multiple epitopes across the protein .

  • Mouse polyclonal antibodies raised against full-length protein offer broad epitope recognition .

  • Discrepancies may result from differential accessibility of epitopes due to protein conformation or interactions.

• Isoform-Specific Recognition:

  • EIF4ENIF1 has multiple transcript variants encoding different isoforms .

  • Verify whether antibodies target isoform-specific regions versus common domains.

  • Cross-reference with expected isoform expression in your experimental system.

• Post-Translational Modification Interference:

  • EIF4ENIF1 exhibits significant mobility differences in Western blots (calculated 88-108 kDa vs. observed 178 kDa) .

  • This suggests extensive post-translational modifications that may mask epitopes.

  • Different antibodies may have variable sensitivity to phosphorylation, ubiquitination, or other modifications.

• Technical Protocol Optimization:

  • Modify fixation conditions for immunofluorescence to expose potentially masked epitopes.

  • Adjust denaturation conditions for Western blotting to account for different antibody requirements.

  • Optimize antibody concentrations independently for each antibody.

• Biological Context Considerations:

  • EIF4ENIF1 functions in nucleocytoplasmic shuttling with context-dependent localization .

  • Different antibodies may preferentially detect nuclear versus cytoplasmic populations.

  • Cell cycle stage or stress conditions may affect epitope accessibility.

• Validation with Orthogonal Methods:

  • Employ mass spectrometry to verify protein identity independent of antibody recognition.

  • Use genetic approaches (CRISPR/Cas9) to tag endogenous protein for antibody-independent detection.

What emerging applications of EIF4ENIF1 antibodies might advance understanding of mRNA metabolism disorders?

EIF4ENIF1 antibodies present significant opportunities for advancing research into mRNA metabolism disorders through several innovative applications:

• Translational Regulation in Neurodevelopmental Disorders:

  • Given EIF4ENIF1's role in translation repression and its relationship to eIF4E (implicated in autism spectrum disorders), antibodies can help map dysregulated translation pathways.

  • Methodological approach: Comparative immunoprecipitation studies of EIF4ENIF1-bound mRNAs in neuronal models of developmental disorders versus controls.

• Connection to Fragile X-Associated Primary Ovarian Insufficiency:

  • Research suggests potential mechanistic parallels between EIF4ENIF1-associated POI and fragile X-associated primary ovarian insufficiency, both possibly involving mRNA toxicity .

  • Experimental design: Use EIF4ENIF1 antibodies to investigate potential interactions with FMRP (Fragile X Mental Retardation Protein) in ovarian tissue.

• Cancer Research Applications:

  • eIF4E plays a role in oncogenesis, with overexpression leading to malignant transformation .

  • Research opportunity: Apply EIF4ENIF1 antibodies to investigate how altered EIF4ENIF1-eIF4E interactions may contribute to dysregulated translation in cancer cells.

  • While no cancers have been reported in carriers of EIF4ENIF1 mutations in the studied family, the mechanistic connection warrants investigation .

• Stress Granule Dynamics in Neurodegeneration:

  • As a regulator of mRNA fate, EIF4ENIF1 may influence stress granule formation relevant to neurodegenerative disorders.

  • Methodology: Use immunofluorescence with EIF4ENIF1 antibodies to track stress granule composition and dynamics in models of neurodegeneration.

• Therapeutic Target Validation:

  • EIF4ENIF1 antibodies can help validate the protein as a potential therapeutic target in disorders of aberrant mRNA stability or translation.

  • Approach: Use antibodies to assess the effects of small molecule modulators of EIF4ENIF1 function on downstream translation pathways.

These emerging applications leverage EIF4ENIF1 antibodies to bridge fundamental research on mRNA metabolism with clinical understanding of associated disorders, potentially opening new avenues for diagnostic and therapeutic development .

How might comparative studies of EIF4ENIF1 across species inform reproductive biology research?

Comparative studies of EIF4ENIF1 across species offer unique insights into evolutionary conservation of reproductive mechanisms and translational control in germ cells:

• Evolutionary Conservation Analysis:

  • Murine Clast4 protein shows 89% homology to human EIF4ENIF1, while Drosophila Cup protein contains highly homologous eIF4E binding motifs .

  • Research approach: Use cross-reactive or species-specific EIF4ENIF1 antibodies to compare protein localization, interactome, and function across species.

  • This may reveal fundamental mechanisms conserved throughout evolution versus species-specific adaptations.

• Reproductive System Comparative Functions:

  • In Drosophila, Cup protein is found only in oocytes and is required for proper accumulation and localization of eIF4E to the posterior cytoplasm of developing oocytes .

  • Similar to human findings, Cup mutations result in abnormal oocyte growth and disruption of meiotic chromosome segregation .

  • Methodological strategy: Apply immunohistochemistry and immunofluorescence with EIF4ENIF1 antibodies to compare protein distribution patterns in reproductive tissues across species.

• Male Fertility Mechanisms:

  • While no male fertility phenotype was observed in the human family with EIF4ENIF1 mutation, Drosophila eIF4E-3 (testis-specific) is essential for male fertility .

  • Research opportunity: Investigate potential interactions between EIF4ENIF1/Cup homologues and testis-specific translation factors across species using co-immunoprecipitation with appropriate antibodies.

  • This could clarify whether the role of EIF4ENIF1 in fertility shows sexual dimorphism across species.

• Developmental Timing Regulation:

  • Compare how EIF4ENIF1/Cup/Clast4 regulate the temporal control of translation during oocyte development across species.

  • Experimental approach: Use immunoprecipitation with EIF4ENIF1 antibodies to identify conserved versus species-specific mRNA targets in oocytes.

• Translational Control Mechanisms:

  • Investigate whether mechanisms of translational repression by EIF4ENIF1/Cup/Clast4 are conserved across species.

  • Methodology: Compare the composition of EIF4ENIF1-containing ribonucleoprotein complexes across species using antibody-based pulldowns followed by proteomic analysis.

These comparative approaches can reveal evolutionarily conserved mechanisms of translational control in reproductive biology, potentially identifying fundamental pathways that could inform human reproductive health research and fertility treatments .