EIF5A4 Antibody

What is EIF5A and why is it significant in cellular research?

EIF5A (eukaryotic translation initiation factor 5A) is a highly conserved protein that plays essential roles in both translation initiation and elongation processes. Despite its initial identification as a translation initiation factor, current research has established its critical function in the elongation step of translation, particularly in the formation of peptidyl bonds. EIF5A has gained significant research interest because it is the only protein known to contain the amino acid residue hypusine, which is formed through a specific post-translational modification of a lysine residue . This unique modification is essential for EIF5A function and cell viability, making it an important target for various cellular and molecular studies. Additionally, the overexpression of EIF5A has been linked to the development of various cancers, suggesting its potential as a therapeutic target .

What experimental applications are suitable for EIF5A antibodies?

EIF5A antibodies have been validated for multiple experimental applications, with specific protocols and dilutions established for optimal results:

When selecting an application, researchers should consider the specific question being addressed and the sensitivity required. Western blot is particularly useful for detecting and quantifying EIF5A protein levels, while IHC and IF provide valuable information about cellular localization and expression patterns across different cell types and tissues. Each application requires specific optimization for the particular experimental system being used .

How should researchers validate the specificity of EIF5A antibodies?

Validating antibody specificity is crucial for ensuring reliable experimental results. For EIF5A antibodies, multiple approaches should be employed:

• Molecular weight verification: Confirm that the observed molecular weight matches the expected size for EIF5A (approximately 18 kDa) .

• Knockdown/knockout controls: Test the antibody in systems where EIF5A has been knocked down or knocked out to verify the absence or reduction of signal. Published literature has documented the use of these approaches for EIF5A antibody validation .

• Positive control samples: Include cells or tissues known to express EIF5A, such as HeLa cells, NIH/3T3 cells, or human brain tissue .

• Peptide competition assays: Pre-incubate the antibody with the immunogen peptide to demonstrate signal reduction, confirming specificity.

• Multiple antibody comparison: When possible, compare results using antibodies generated against different epitopes of EIF5A to confirm consistency in detection patterns.

How can researchers differentiate between hypusinated and non-hypusinated forms of EIF5A?

The hypusination of EIF5A is a critical post-translational modification that significantly impacts its functionality. This unique modification involves a two-step enzymatic process catalyzed by Deoxyhypusine Synthase (DHPS) and Deoxyhypusine Hydroxylase (DOHH) . To differentiate between hypusinated and non-hypusinated forms of EIF5A:

• Hypusine-specific antibodies: Some specialized antibodies are designed to specifically recognize the hypusinated form of EIF5A. These can be used alongside pan-EIF5A antibodies to determine the ratio of modified to unmodified protein.

• 2D gel electrophoresis: This approach separates proteins based on both isoelectric point and molecular weight, allowing for the detection of post-translationally modified variants that may have subtle charge differences.

• Mass spectrometry analysis: For definitive identification and quantification of hypusination, mass spectrometry can be used to detect the specific mass shift associated with the hypusine modification.

• Inhibitor studies: Treatment with DHPS inhibitors (such as GC7) or DOHH inhibitors can be used to manipulate hypusination levels, allowing researchers to study the functional consequences of this modification and confirm antibody specificity .

How does EIF5A subcellular localization impact experimental design and data interpretation?

EIF5A demonstrates complex subcellular distribution patterns that are functionally significant. While primarily associated with translation machinery in the cytoplasm, EIF5A has also been found in the nucleus, mitochondria, and endoplasmic reticulum (ER) . This diverse localization profile has important implications for experimental design:

• Acetylation-dependent nuclear translocation: EIF5A can be acetylated by p300/CBP-associated factor (PCAF), which leads to its translocation to the nucleus and deactivation of its translation-related functions . Researchers should consider using acetylation-specific antibodies or acetylation inhibitors when studying nuclear EIF5A.

• Mitochondrial localization: EIF5A's presence in mitochondria is associated with regulation of mitochondrial activity and apoptotic processes . When studying mitochondrial functions, subcellular fractionation and co-localization studies with mitochondrial markers should be considered.

• ER association: EIF5A's presence in the ER is linked to maintenance of ER integrity and control of the unfolded protein response (UPR) . Studies focusing on ER stress should include analysis of EIF5A localization and function.

• Immunofluorescence controls: When performing IF/ICC experiments, appropriate markers for each subcellular compartment should be included to accurately interpret EIF5A localization patterns.

What are the critical considerations for studying EIF5A in cancer research models?

Given the association between EIF5A overexpression and cancer development, this protein has emerged as an important subject in cancer research . When designing cancer-related studies involving EIF5A antibodies:

• Cancer-specific expression profiling: Different cancer types may show variable patterns of EIF5A expression and hypusination. Comprehensive profiling across multiple cancer cell lines and patient-derived samples is recommended, with appropriate normal tissue controls.

• Therapeutic targeting considerations: When studying EIF5A as a therapeutic target, researchers should evaluate both total EIF5A levels and the degree of hypusination, as both factors influence cancer progression .

• Multiple myeloma, B-Cell lymphoma, and neuroblastoma models: These cancer types have been specifically linked to EIF5A dysregulation and represent particularly relevant model systems for studying EIF5A function and therapeutic targeting .

• Interplay with DHPS and DOHH: The enzymes responsible for hypusination (DHPS and DOHH) are also potential therapeutic targets in cancer. Studies should consider the relationship between these enzymes and EIF5A levels/activity .

• Careful control selection: When using EIF5A antibodies in cancer research, matched normal tissues or non-transformed cell lines should be included as controls to establish baseline expression and localization patterns.

What sample preparation protocols optimize EIF5A antibody performance in various applications?

Proper sample preparation is critical for obtaining reliable results with EIF5A antibodies. Based on validated protocols:

For Western Blot:

• Use buffer containing protease inhibitors to prevent degradation of EIF5A during extraction.

• Include phosphatase inhibitors when studying potential phosphorylation events.

• Denature samples completely (95-100°C for 5 minutes) in reducing sample buffer to ensure proper denaturation.

• Load appropriate protein amounts (typically 10-30 μg of total protein) for optimal detection.

For Immunohistochemistry:

• For formalin-fixed, paraffin-embedded tissues, antigen retrieval using TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may also be effective .

• Optimize blocking conditions to minimize background signal while maintaining specific staining.

• Consider the inclusion of signal amplification methods for detecting low-abundance expression.

For Immunofluorescence:

• Fixation method significantly impacts epitope accessibility; both paraformaldehyde and methanol fixation protocols should be tested for optimal results.

• Permeabilization conditions should be optimized for the specific cell type being studied.

• Include appropriate controls for autofluorescence and non-specific binding.

How should researchers address technical challenges in EIF5A antibody experiments?

When working with EIF5A antibodies, several technical challenges may arise that require methodological adjustments:

• Background reduction: If high background is observed in Western blots or immunostaining:

  • Increase blocking time or blocking agent concentration

  • Optimize antibody dilution (testing a range from 1:5000 to 1:50000 for WB and 1:50 to 1:500 for IHC/IF)

  • Include additional washing steps with increased detergent concentration

  • For monoclonal antibodies, try different clones if available

• Signal enhancement: For detection of low-abundance EIF5A:

  • Consider using enhanced chemiluminescence (ECL) substrates with higher sensitivity for Western blots

  • Implement biotin-streptavidin amplification systems for immunostaining

  • Optimize exposure times for imaging

• Cross-reactivity assessment: To ensure specificity:

  • Test the antibody across multiple species if cross-species reactivity is claimed

  • Include negative control tissues or cell lines with low/no EIF5A expression

  • For polyclonal antibodies, consider affinity purification against the immunogen

What controls are essential for interpreting EIF5A antibody data?

Proper experimental controls are fundamental for accurate data interpretation:

• Positive controls: Include samples known to express EIF5A, such as:

  • HeLa cells, NIH/3T3 cells, and PC-3 cells for Western blot

  • Mouse testis tissue for immunohistochemistry

  • HeLa cells or HEK-293 cells for immunofluorescence

• Negative controls:

  • Primary antibody omission to assess secondary antibody specificity

  • Isotype controls (especially for monoclonal antibodies) to evaluate non-specific binding

  • EIF5A-knockdown or knockout samples when available

• Loading controls for Western blot:

  • Use housekeeping proteins (β-actin, GAPDH, tubulin) to normalize EIF5A expression

  • Consider using total protein normalization methods for more accurate quantification

• Replication and statistical validation:

  • Perform at least three independent biological replicates

  • Apply appropriate statistical tests based on data distribution

  • Report variability measures (standard deviation, standard error) alongside means

How can EIF5A antibodies be utilized in studying stress response mechanisms?

EIF5A is involved in cellular stress responses, particularly through its role in the unfolded protein response (UPR) and maintenance of ER integrity . When investigating stress responses:

• Stress induction protocols: Researchers can induce various stressors (oxidative stress, ER stress, nutrient deprivation) and monitor changes in EIF5A expression, localization, and post-translational modifications using specific antibodies.

• Co-localization studies: Combine EIF5A antibodies with markers for stress granules, processing bodies, or UPR components to examine potential interactions during stress conditions.

• Time-course experiments: Monitor EIF5A dynamics during acute and chronic stress exposure, correlating changes with cellular outcomes (adaptation, senescence, apoptosis).

• Stress-specific post-translational modifications: Investigate whether stress conditions alter the hypusination or acetylation status of EIF5A, potentially affecting its function and localization.

What emerging techniques can enhance EIF5A antibody-based research?

Several cutting-edge methodologies can be integrated with EIF5A antibody applications to advance research capabilities:

• Proximity ligation assays (PLA): This technique can detect protein-protein interactions involving EIF5A with higher sensitivity than traditional co-immunoprecipitation, providing spatial information about where in the cell these interactions occur.

• Single-cell analysis: Combining EIF5A antibodies with single-cell technologies can reveal cell-to-cell variability in EIF5A expression and modification states within heterogeneous populations.

• Super-resolution microscopy: Techniques such as STORM, PALM, or STED microscopy can provide nanoscale resolution of EIF5A localization, potentially revealing previously undetectable distribution patterns.

• CRISPR-Cas9 gene editing: Generation of endogenously tagged EIF5A allows for live-cell imaging and functional studies with reduced concerns about antibody specificity.

• Ribosome profiling: Combining ribosome profiling with EIF5A modulation (knockdown, overexpression, hypusination inhibition) can provide insights into how EIF5A regulates translation at specific mRNAs.