EIF5A5 Antibody

What is EIF5A and why is it significant in molecular biology research?

EIF5A (Eukaryotic translation initiation factor 5A) is a highly conserved protein that plays crucial roles in translation elongation rather than initiation as initially thought. Despite its name, EIF5A functions primarily as an elongation factor that facilitates translation of specific mRNA sequences, particularly those containing consecutive proline codons (polyproline motifs). It is the only known protein containing the unique amino acid hypusine, formed through post-translational modification of a specific lysine residue .

Two paralogous genes encode distinct EIF5A isoforms: EIF5A1, which is abundantly expressed in most cells and tissues, and EIF5A2, which is rare in normal tissues but frequently overexpressed in various malignancies. Both isoforms require hypusination for biological activity . The differential distribution and sequence differences between these isoforms are particularly relevant for cancer research and therapeutic development.

What types of EIF5A antibodies are currently available for research?

Based on current research resources, EIF5A antibodies are available in several formats:

Researchers should select antibodies based on their specific experimental requirements, including target isoform (EIF5A1 vs. EIF5A2), species reactivity (human, mouse, rat), and intended application .

How should researchers validate EIF5A antibody specificity?

Validating antibody specificity is critical for obtaining reliable research data. For EIF5A antibodies, a comprehensive validation approach should include:

• Western blot analysis using positive control cell lines known to express EIF5A (e.g., HeLa, NIH/3T3, PC-3 cells)

• Comparison with a knockout or knockdown control (siRNA against EIF5A)

• Peptide competition assays to confirm epitope specificity

• Cross-reactivity testing if working with multiple species

• Comparison of detection patterns across multiple antibodies targeting different epitopes

For distinguishing between EIF5A1 and EIF5A2 isoforms, particular attention must be paid to antibody epitope regions, as these isoforms share significant sequence homology .

What are the optimal conditions for using EIF5A antibodies in Western blotting?

For successful Western blot detection of EIF5A:

• Sample preparation: Standard cell or tissue lysis protocols are generally effective; phosphatase inhibitors should be included if phosphorylation status is relevant

• Protein loading: 10-30 μg of total protein is typically sufficient

• Separation: 12-15% SDS-PAGE gels are recommended due to EIF5A's relatively small size (18 kDa)

• Transfer: Standard wet or semi-dry transfer protocols; PVDF membranes may provide better retention

• Blocking: 5% non-fat milk or BSA in TBST (1 hour at room temperature)

• Primary antibody: Dilutions typically range from 1:1000-1:5000, though some antibodies may be effective at much higher dilutions (1:50000)

• Incubation time: Overnight at 4°C generally yields optimal results

• Detection system: Both chemiluminescence and fluorescence-based detection systems are suitable

Expected molecular weights: EIF5A1 and EIF5A2 appear at approximately 17-18 kDa. Higher molecular weight bands may indicate post-translational modifications or oligomerization .

What protocols yield optimal results for immunohistochemistry with EIF5A antibodies?

For effective IHC staining of EIF5A in tissue sections:

• Fixation: Standard formalin fixation and paraffin embedding is compatible with most available EIF5A antibodies

• Antigen retrieval:

  • Heat-induced epitope retrieval using TE buffer (pH 9.0) is recommended for many EIF5A antibodies

  • Citrate buffer (pH 6.0) serves as an alternative retrieval method

• Blocking: 1-2% normal serum from the species of the secondary antibody

• Primary antibody: Typical dilutions range from 1:50 to 1:500 depending on the specific antibody

• Incubation: 1 hour at room temperature or overnight at 4°C

• Detection: Standard polymer-based or ABC detection systems

• Counterstaining: Hematoxylin provides good contrast

Key recommendations:

• Include both positive control tissues (e.g., liver for EIF5A1, tumor samples for EIF5A2) and negative controls

• For proper interpretation, note that EIF5A1 is predominantly cytoplasmic, while subcellular localization may change based on its hypusination status

How should researchers approach the detection of hypusinated versus non-hypusinated EIF5A?

Distinguishing between hypusinated and non-hypusinated forms of EIF5A presents technical challenges:

• Commercial options:

  • Specialized antibodies targeting hypusinated EIF5A are available but limited

  • These require extensive validation due to the subtle nature of this modification

• Alternative approaches:

  • 2D gel electrophoresis can separate the modified forms based on charge differences

  • Mass spectrometry following enrichment of the modified protein

  • Indirect assessment using inhibitors of hypusination (e.g., GC7, a DHS inhibitor) to create control samples with reduced hypusination

• Experimental considerations:

  • Subcellular localization studies can provide indirect evidence, as hypusinated EIF5A is predominantly cytoplasmic while non-hypusinated precursor is found in both nucleus and cytoplasm

  • Western blotting with specialized hypusine-specific antibodies requires careful optimization of conditions

How can researchers effectively use EIF5A antibodies to study its role in cancer?

EIF5A's role in cancer, particularly the differential expression of its isoforms, can be investigated using several approaches:

• Expression profiling:

  • IHC analysis of tumor tissue microarrays to correlate EIF5A1/EIF5A2 expression with clinicopathological parameters

  • Western blot comparison between tumor and adjacent normal tissues

  • Combine with markers like Ki-67 to assess correlation with proliferative index

• Prognostic assessment:

  • Quantitative analysis of EIF5A expression levels in correlation with patient survival data

  • In lung adenocarcinoma, higher EIF5A1 expression correlates with poorer prognosis

  • EIF5A2 amplification occurs in 9% of lung adenocarcinoma cases and correlates with poor outcomes

• Functional studies:

  • Knockdown/knockout approaches using siRNA or CRISPR-Cas9

  • Overexpression of wild-type or mutant EIF5A

  • Combined with phenotypic assays for proliferation, migration, and invasion

• Pathway analysis:

  • Co-immunoprecipitation to identify EIF5A interaction partners

  • Analysis of downstream effects on translation of polyproline-containing proteins

  • Connection to TGF-β signaling pathways in cancer progression

What considerations are important when studying EIF5A in immune cell function?

EIF5A plays critical roles in immune cell function, particularly in T cells and NK cells:

• Experimental approach for NK cells:

  • Flow cytometry using EIF5A antibodies can determine expression levels in NK cell subpopulations

  • Confocal microscopy reveals subcellular localization of EIF5A in NK cells (predominantly cytoplasmic in untreated cells)

  • Inhibition studies using GC7 (DHS inhibitor) can elucidate the role of hypusinated EIF5A in NK cell function

• T cell research considerations:

  • EIF5A is essential for long-term survival of effector CD8+ T cells

  • Its function is regulated dynamically in naïve CD8+ T cells upon activation

  • EIF5A is critical for IFNγ production and less acutely for TNF production and cytotoxic functions

• Study design elements:

  • Include time course experiments to capture dynamic regulation of EIF5A

  • Combine with functional assays (proliferation, cytokine production, cytotoxicity)

  • Consider both direct effects on immune cells and indirect effects on immune microenvironment

How can puromycin incorporation assays be combined with EIF5A antibodies to study translation dynamics?

Puromycin incorporation assays provide valuable insights into active translation sites when combined with EIF5A immunostaining:

• Experimental protocol:

  • Treat cells with puromycin (typically 10 μg/ml for 5-10 minutes)

  • Fix cells and perform immunofluorescence using anti-puromycin antibody and anti-EIF5A antibody

  • Perform confocal microscopy to assess colocalization

• Data interpretation:

  • Colocalization of EIF5A with puromycin indicates sites of active translation

  • In H1395-EIF5A2 cells treated with TGF-β1, increased puromycin incorporation correlates with higher translational activity

  • Positional colocalization of Flag-EIF5A2 with puromycin confirms active translation sites where EIF5A2 may facilitate translation of polyproline-containing proteins

• Advanced applications:

  • Combine with ribosome profiling to identify specific mRNAs being translated

  • Use cycloheximide chase experiments to assess impact on protein stability

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to study EIF5A dynamics at translation sites

How do EIF5A1 and EIF5A2 isoforms differ functionally in cancer progression?

Research indicates distinct functional roles for EIF5A isoforms in cancer:

• Differential effects on cell phenotypes:

  • EIF5A1 depletion increased cell proliferation by 14% and 2% in A549 and H1395 lung cancer cells, respectively

  • EIF5A2 depletion decreased proliferation by 38% in A549 and 20% in H1395 cells

  • Both isoforms affect migration, with EIF5A2 depletion having more pronounced effects

• Cytoskeletal organization:

  • EIF5A2 depletion causes more severe disruption of actin cytoskeleton than EIF5A1 depletion

  • Effects include broken and disorganized actin fibers and cytoplasmic aggregates

  • This likely relates to EIF5A's role in translating formin proteins, which contain polyproline domains

• Tumor growth and invasion:

  • H1395 cells overexpressing EIF5A2 showed faster initial tumor growth in mouse xenograft models

  • EIF5A2-overexpressing cells formed larger aggregates in lung tissue, suggesting enhanced invasive capacity

• Gene amplification patterns:

  • 9% of lung adenocarcinoma samples show amplification and high mRNA levels of EIF5A2

  • 5% show high levels of mRNA and deep deletion for EIF5A1 gene

What are the current methodological challenges in studying EIF5A's elongation function?

Investigating EIF5A's role in translation elongation presents several technical challenges:

• Ribosome stalling detection:

  • Ribosome profiling can identify sites of ribosome stalling at polyproline motifs

  • Requires specialized bioinformatic analyses to distinguish genuine stalling from technical artifacts

  • Should include EIF5A depletion or hypusination inhibition conditions as controls

• Isolating client mRNAs:

  • Identifying mRNAs specifically dependent on EIF5A for efficient translation

  • Approaches include polysome profiling combined with RNA-seq in EIF5A-depleted vs. control cells

  • Nascent peptide sequencing can identify proteins whose production depends on functional EIF5A

• Temporal dynamics:

  • Translation is a rapid process, requiring techniques with high temporal resolution

  • Single-molecule imaging approaches can provide insights but require specialized equipment

  • Synchronization of cells may help capture specific translation events

• Distinguishing direct vs. indirect effects:

  • EIF5A affects translation of proteins that themselves regulate other processes

  • Careful experimental design with appropriate controls is necessary

  • Acute inhibition or depletion approaches may help distinguish primary from secondary effects

What therapeutic strategies targeting EIF5A are being explored, and how can antibodies facilitate this research?

Several therapeutic approaches targeting EIF5A are under investigation, with antibodies playing crucial roles in development and validation:

• Inhibition of hypusination pathway:

  • DHS (deoxyhypusine synthase) inhibitors: GC7 is widely used in research

  • DOHH (deoxyhypusine hydroxylase) inhibitors

  • Antibodies can assess efficacy by measuring changes in hypusinated vs. non-hypusinated EIF5A levels

• Gene expression modulation:

  • siRNA/shRNA approaches to reduce EIF5A expression

  • microRNAs directed against DHS or DOHH

  • Antibodies enable verification of knockdown efficiency

• Overexpression of dominant-negative mutants:

  • Expression of EIF5A mutants that cannot be hypusinated

  • Antibodies help distinguish between endogenous and exogenous protein expression

• Combination therapies:

  • EIF5A-targeting approaches combined with conventional chemotherapeutics (imatinib, 5-fluorouracil, cisplatin)

  • Antibodies facilitate assessment of synergistic effects on target pathways

• Translation monitoring:

  • Antibodies against EIF5A and its client proteins can monitor therapeutic efficacy

  • Particularly relevant for proteins involved in cell migration, invasion, and proliferation

How can researchers address non-specific binding issues with EIF5A antibodies?

Non-specific binding presents challenges in EIF5A detection. Researchers can implement several strategies:

• Optimization of blocking conditions:

  • Test different blocking agents (BSA, non-fat milk, normal serum)

  • Extend blocking time to 1-2 hours at room temperature

  • Include 0.1-0.3% Triton X-100 or Tween-20 in blocking solution

• Antibody dilution optimization:

  • Perform a dilution series to determine optimal concentration

  • For Western blots, some antibodies require significant dilution (1:5000-1:50000)

  • For IHC/IF, typically 1:50-1:500 range is appropriate

• Cross-adsorption techniques:

  • Pre-adsorb antibody with cell/tissue lysates from species with high homology

  • Use recombinant protein for pre-adsorption if available

• Alternative antibody selection:

  • If persistent non-specific binding occurs, consider antibodies targeting different epitopes

  • Monoclonal antibodies may provide higher specificity than polyclonal antibodies in some applications

• Stringent washing protocols:

  • Increase wash duration and buffer volume

  • Consider higher salt concentration in wash buffers (up to 500 mM NaCl)

  • Add 0.05-0.1% SDS to wash buffer for Western blot applications

What controls are essential for experiments investigating EIF5A hypusination?

When studying EIF5A hypusination, appropriate controls are critical:

• Positive controls:

  • Proliferating cell lines known to have high levels of hypusinated EIF5A (e.g., HeLa cells)

  • Recombinant hypusinated EIF5A protein (if available)

• Negative controls:

  • Cells treated with hypusination inhibitors (e.g., GC7)

  • Cells expressing lysine-to-arginine mutant EIF5A (cannot be hypusinated)

  • Serum-starved cells (reduced hypusination)

• Specificity controls:

  • Peptide competition assays with hypusinated and non-hypusinated peptides

  • Knockdown/knockout cells for antibody validation

• Comparative analysis:

  • Track cytoplasmic vs. nuclear localization as indirect measure of hypusination status

  • Non-hypusinated EIF5A shows increased nuclear localization

  • Include assessment of cellular morphology (crescent-shaped chromatin aggregates may appear with hypusination inhibition)

How should researchers interpret conflicting data between EIF5A antibodies from different sources?

When faced with inconsistent results using different EIF5A antibodies, consider:

• Systematic comparison approach:

  • Test multiple antibodies under identical conditions

  • Document epitope regions, host species, and clonality of each antibody

  • Create a comparison table of results across applications

• Epitope availability factors:

  • Different fixation methods may mask certain epitopes

  • Post-translational modifications may affect antibody binding

  • Protein-protein interactions could block epitope access

• Validation strategies:

  • Genetic approaches (siRNA, CRISPR) to validate specificity

  • Recombinant protein expression as positive control

  • Mass spectrometry to confirm protein identity

• Isoform specificity considerations:

  • Determine whether antibodies distinguish between EIF5A1 and EIF5A2

  • Map epitopes to regions of sequence divergence between isoforms

  • Consider tissue-specific expression patterns (EIF5A2 is rare in normal tissues)

• Reporting recommendations:

  • Clearly document antibody source, catalog number, and lot in publications

  • Include detailed methods for reproducibility

  • Acknowledge limitations of specific antibodies in interpretations