EIF4ENIF1 Antibody, HRP conjugated

What is EIF4ENIF1 and why is it significant in translation regulation research?

EIF4ENIF1 (Eukaryotic Translation Initiation Factor 4E Nuclear Import Factor 1) functions as a nucleocytoplasmic shuttle protein for the translation initiation factor eIF4E. It interacts with the importin alpha-beta complex to mediate nuclear import of eIF4E. While predominantly cytoplasmic, its own nuclear import is regulated by specific nuclear localization signals and nuclear export signals. Multiple transcript variants encoding different isoforms have been found for this gene . The importance of studying EIF4ENIF1 lies in understanding the regulation of eIF4E localization, which is critical for controlling cap-dependent translation initiation, a process frequently dysregulated in cancer and other diseases .

What experimental applications is the EIF4ENIF1 Antibody (HRP conjugated) validated for?

The EIF4ENIF1 Antibody (HRP conjugated) has been specifically validated for ELISA applications with high sensitivity and specificity. The horseradish peroxidase (HRP) conjugation enables direct detection without secondary antibodies, making it particularly valuable for quantitative ELISAs and protein detection assays where minimizing background signal is essential . While primarily validated for ELISA, researchers should optimize conditions when adapting this antibody for other applications such as immunohistochemistry or Western blotting.

What are the optimal storage and handling conditions for maintaining antibody activity?

To maintain optimal activity of the EIF4ENIF1 Antibody (HRP conjugated), the following protocol is recommended:

• Upon receipt, aliquot the antibody into smaller volumes to minimize freeze-thaw cycles

• Store aliquots at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles as this significantly reduces antibody activity

• Protect from light exposure due to the light-sensitive nature of the HRP conjugate

• Store in the provided buffer (0.01M PBS, pH 7.4, with 0.03% Proclin-300 and 50% Glycerol)

Following these guidelines will help maintain >95% antibody purity and activity for the expected shelf life.

How should researchers determine the optimal antibody dilution for specific experimental applications?

When optimizing dilution factors for the EIF4ENIF1 Antibody (HRP conjugated), researchers should implement a systematic titration approach:

• Begin with a broad dilution range (e.g., 1:500, 1:1000, 1:2000, 1:5000)

• Conduct preliminary experiments using positive controls (tissues or cell lines known to express EIF4ENIF1)

• Include negative controls (knockout tissues or cell lines) when available

• Assess signal-to-noise ratio at each dilution

• Calculate signal specificity by comparing signal intensity between positive and negative controls

• Select the dilution that provides maximum specific signal while minimizing background noise

This methodical approach avoids wastage of valuable antibody while ensuring optimal results. The manufacturer notes that "optimal dilutions/concentrations should be determined by the end user" as these parameters can vary significantly depending on sample type and detection method .

What sample preparation protocols optimize EIF4ENIF1 detection in different experimental systems?

Sample preparation significantly impacts antibody performance. For optimal EIF4ENIF1 detection:

For cell lysates:

• Lyse cells in RIPA buffer supplemented with protease inhibitors

• Include phosphatase inhibitors if phosphorylation status is relevant

• Perform lysis on ice (4°C) for 30 minutes with periodic vortexing

• Centrifuge at 14,000 × g for 15 minutes at 4°C

• Collect supernatant and determine protein concentration

For tissue samples:

• Homogenize fresh tissue in ice-cold lysis buffer (1:10 w/v)

• Use mechanical disruption followed by sonication

• Centrifuge at 14,000 × g for 20 minutes at 4°C

• Collect supernatant and filter through a 0.45 μm filter

• Determine protein concentration before proceeding with analysis

These protocols help preserve protein integrity and epitope accessibility, critical factors for antibody recognition.

How can the EIF4ENIF1 Antibody be applied to investigate translational control in breast cancer models?

Recent research demonstrates the critical role of the eIF4E/eIF4A axis in breast cancer progression. When investigating translational control mechanisms in breast cancer models, researchers can implement the following protocol:

• Use the EIF4ENIF1 Antibody to track changes in eIF4E nuclear import/export dynamics following treatment with eIF4A inhibitors like zotatifin

• Compare EIF4ENIF1 localization in hormone-dependent versus hormone-independent breast cancer cells

• Correlate EIF4ENIF1 expression levels with ER (estrogen receptor) translation rates

• Combine with methionine analog labeling (e.g., L-Azidohomoalanine) to measure de novo protein synthesis rates

• Assess how modulation of the EIF4ENIF1-eIF4E interaction affects translation of specific mRNAs implicated in cancer progression

This approach provides mechanistic insights into how eIF4A inhibition reduces ER expression and suppresses ER-dependent transcription in breast cancer models, potentially revealing new therapeutic targets .

What is the relationship between EIF4ENIF1 function and mRNA selectivity in oncogenic transformation?

The relationship between EIF4ENIF1 function and mRNA selectivity represents a critical area for cancer research:

• EIF4ENIF1 regulates nuclear-cytoplasmic shuttling of eIF4E, affecting availability for cap-dependent translation

• During oncogenic transformation, eIF4E levels and phosphorylation increase, altering the translational landscape

• Specifically, a 50% reduction in eIF4E expression significantly impedes cellular transformation without affecting normal development

• This suggests cancer cells require excess eIF4E levels beyond what's needed for normal physiology

• Genome-wide translational profiling reveals that eIF4E dose is essential for translating mRNAs with unique 5′UTR signatures, particularly those regulating reactive oxygen species (ROS) that fuel transformation

These findings indicate that monitoring EIF4ENIF1-mediated eIF4E localization provides insights into how cancer cells hijack translation machinery to support tumorigenesis.

How can researchers design experiments to distinguish between direct and indirect effects of EIF4ENIF1 modulation?

To differentiate direct from indirect effects of EIF4ENIF1 modulation, implement the following experimental design:

Direct vs. Indirect Effects Experimental Framework:

This comprehensive approach helps distinguish primary effects of EIF4ENIF1 modulation from secondary adaptive responses, providing clearer mechanistic insights into translation regulation pathways.

What are the critical considerations when investigating EIF4ENIF1 in stress response and cancer cell survival?

When investigating EIF4ENIF1 in stress responses and cancer cell survival, researchers should address these critical considerations:

• Microenvironmental context: Assess EIF4ENIF1 function under various stress conditions (hypoxia, nutrient deprivation, oxidative stress) that mimic tumor microenvironments

• Temporal dynamics: Monitor EIF4ENIF1 localization and activity at multiple time points, as stress responses often involve biphasic or multiphasic regulation

• Metabolic dependencies: Integrate measurements of reactive oxygen species (ROS) levels, as eIF4E dose is essential for translating mRNAs regulating ROS that fuel transformation and cancer cell survival

• Therapeutic implications: Evaluate how modulation of EIF4ENIF1 affects response to standard therapies, particularly in combination with eIF4A inhibitors like zotatifin which have shown promise in clinical trials (NCT04092673)

• Pathway redundancies: Assess compensatory mechanisms that may activate when EIF4ENIF1-mediated regulation is disrupted

Understanding these considerations provides a comprehensive framework for investigating how cancer cells manipulate translation machinery through EIF4ENIF1 to survive stress conditions.

What are common technical challenges when using EIF4ENIF1 Antibody (HRP conjugated) and how can they be addressed?

Researchers frequently encounter these challenges when using EIF4ENIF1 Antibody (HRP conjugated):

Implementing these troubleshooting strategies ensures reliable and reproducible results when working with EIF4ENIF1 Antibody (HRP conjugated).

How can researchers validate antibody specificity for EIF4ENIF1 detection in their experimental system?

Rigorous validation of antibody specificity is essential for reliable research outcomes. For EIF4ENIF1 Antibody, implement this validation pipeline:

• Genetic validation:

  • Use CRISPR/Cas9 knockout cells or siRNA knockdown samples

  • Compare signal in wild-type vs. EIF4ENIF1-depleted samples

  • Expected result: Significant signal reduction in knockout/knockdown samples

• Peptide competition:

  • Pre-incubate antibody with excess synthetic EIF4ENIF1 peptide

  • Compare signal with and without peptide competition

  • Expected result: Specific signals should be blocked by peptide pre-incubation

• Multiple antibody validation:

  • Use alternative antibodies targeting different EIF4ENIF1 epitopes

  • Compare detection patterns across antibodies

  • Expected result: Consistent detection pattern with multiple antibodies

• Recombinant protein controls:

  • Test antibody against purified recombinant EIF4ENIF1

  • Include dilution series to establish detection limits

  • Expected result: Linear dose-response relationship

• Orthogonal confirmation:

  • Correlate protein detection with mRNA expression (qPCR)

  • Confirm expected subcellular localization pattern

  • Expected result: Protein levels should generally correlate with mRNA expression

This comprehensive validation ensures that experimental observations truly reflect EIF4ENIF1 biology rather than antibody artifacts.

How does EIF4ENIF1 function relate to therapeutic strategies targeting the eIF4F complex in cancer?

The relationship between EIF4ENIF1 function and therapeutic strategies targeting the eIF4F complex offers several clinically relevant insights:

• Synergistic therapeutic potential: Inhibition of eIF4A combined with fulvestrant (an ER degrader) produces synergistic inhibition of ER expression and tumor growth in breast cancer xenograft models. This combination strategy addresses both ER synthesis and degradation simultaneously .

• Clinical translation: Phase I/II clinical trial (NCT04092673) using the eIF4A inhibitor zotatifin in combination with fulvestrant has shown promising results in patients with estrogen receptor-positive metastatic breast cancer, with multiple tumor regressions observed in heavily pre-treated endocrine therapy-resistant patients .

• Mechanistic basis: Targeting EIF4ENIF1 could potentially disrupt nuclear-cytoplasmic shuttling of eIF4E, complementing direct inhibition of eIF4A. This multi-targeted approach to the eIF4F complex may overcome resistance mechanisms observed with single-agent therapies.

• Biomarker potential: EIF4ENIF1 expression levels could serve as predictive biomarkers for response to eIF4F-targeting therapies, helping identify patients most likely to benefit from these approaches.

• Normal tissue toxicity window: Since a 50% reduction in eIF4E is compatible with normal development but significantly impedes cellular transformation, therapies targeting this pathway have a potential therapeutic window that spares normal tissues while affecting cancer cells .

These findings suggest that understanding EIF4ENIF1 biology provides critical insights for developing more effective translation-targeting cancer therapies.

What insights can be gained from studying the relationship between EIF4ENIF1 and mRNA-specific translation regulation?

Studying the relationship between EIF4ENIF1 and mRNA-specific translation regulation reveals fundamental mechanisms of gene expression control:

• 5′UTR signature recognition: Gene sets translationally regulated by transformation are enriched in protein-protein interactions with several functional clusters, including those involved in cell cycle control, signaling, cell-to-cell communication, cell adhesion, and protein homeostasis .

• Cancer-specific vulnerability: Cancer cells appear to hijack eIF4E levels in excess of what's required for normal development to drive a translational program supporting tumorigenesis. This may explain why cancer cells are more sensitive to partial inhibition of this pathway than normal cells .

• Stress response modulation: EIF4ENIF1-mediated control of eIF4E localization may serve as a rapid response mechanism during cellular stress, allowing for quick reprogramming of the translational landscape without changes in transcription.

• Therapeutic target identification: Understanding which mRNAs are most dependent on EIF4ENIF1-regulated processes helps identify potential downstream therapeutic targets that may be more specific than directly targeting the translation machinery.

• Metabolic dependencies: eIF4E dose is essential for translating mRNAs regulating reactive oxygen species (ROS) that fuel transformation and cancer cell survival in vivo, suggesting potential metabolic vulnerabilities that could be therapeutically exploited .

This research direction provides a deeper understanding of how translation regulation contributes to cancer phenotypes and highlights new intervention strategies beyond conventional approaches.