EIF5A Antibody, Biotin conjugated

What is EIF5A and why is it significant in translation studies?

EIF5A is an essential protein that functions globally in translation elongation and termination, not limited to initiation as its name suggests. Recent research has revealed that eIF5A has a much broader role than originally thought, functioning to stimulate translation elongation in many peptide contexts and accelerating the rate of peptidyl-tRNA hydrolysis by eRF1 during termination. This makes eIF5A an obligate translation factor acting on many (if not all) translating ribosomes, which explains its essential nature and high abundance in eukaryotic cells . Studying EIF5A can provide critical insights into fundamental translation mechanisms, especially in resolving ribosome pausing at challenging sequence motifs.

What are the molecular properties of EIF5A protein?

EIF5A is a relatively small protein with the following characteristics:

This 18 kDa protein undergoes a unique post-translational modification called hypusination, which is critical for its function in translation elongation and termination .

What are the validated applications for biotin-conjugated EIF5A antibodies?

Biotin-conjugated EIF5A antibodies have been validated for several experimental techniques:

It's important to note that these ranges are starting points, and each application may require titration for optimal results in your specific experimental system .

What are the advantages of using biotin-conjugated antibodies versus unconjugated ones?

Biotin-conjugated EIF5A antibodies offer several methodological advantages:

• Enhanced sensitivity through avidin/streptavidin amplification systems, which is particularly valuable when detecting low-abundance forms of EIF5A

• Versatility in detection methods, as the biotin tag can be detected using various streptavidin conjugates (HRP, fluorophores, gold particles)

• Compatibility with multiplexing approaches when combined with directly labeled antibodies against other targets

• Signal amplification capabilities, particularly useful for challenging applications like detecting EIF5A in tissues with low expression

• Reduced non-specific background when used with streptavidin detection systems compared to secondary antibody approaches

How should I design experiments to study EIF5A's role in translation elongation?

To investigate EIF5A's function in elongation, consider these methodological approaches:

• Ribosome profiling with EIF5A depletion:

  • Deplete EIF5A using siRNA or CRISPR

  • Confirm depletion efficiency using Western blot

  • Perform ribosome profiling to identify ribosome stalling sites

  • Analyze 5' to 3' ribosome distribution on mRNAs

Research has demonstrated that eIF5A depletion causes a global elongation defect with ribosomes stalling at many sequences, not limited to proline stretches. There is also ribosome accumulation at stop codons and in the 3′ UTR, suggesting a global defect in termination in the absence of eIF5A .

• In vitro translation assays:

  • Set up reconstituted translation systems with and without EIF5A

  • Use reporter mRNAs containing known stalling sequences

  • Measure translation efficiency and peptide synthesis rates

• Polysome analysis:

  • Prepare polysome profiles from control and EIF5A-depleted cells

  • Calculate polysome/monosome ratios (typically increased in EIF5A-depleted cells)

  • Analyze specific mRNA distribution across polysome fractions

What protocol should I follow for immunohistochemistry with biotin-conjugated EIF5A antibodies?

For optimal IHC detection of EIF5A:

• Sample preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Process and embed in paraffin

  • Section at 4-5 μm thickness

• Deparaffinization and rehydration:

  • Xylene: 2 × 5 minutes

  • 100% ethanol: 2 × 3 minutes

  • 95%, 80%, 70% ethanol: 1 × 3 minutes each

  • Rinse in distilled water

• Antigen retrieval (critical step):

  • Recommended method: TE buffer pH 9.0 as specifically suggested for EIF5A

  • Alternative method: Citrate buffer pH 6.0

  • Heat in pressure cooker or microwave until boiling, then 20 minutes at sub-boiling temperature

• Blocking and antibody incubation:

  • Block endogenous peroxidase: 3% H₂O₂ for 10 minutes

  • Block endogenous biotin: Avidin/biotin blocking kit

  • Protein blocking: 5% normal serum for 1 hour

  • Primary antibody: Dilute biotin-conjugated EIF5A antibody 1:50-1:500; incubate overnight at 4°C

  • Detection: Apply streptavidin-HRP for 30 minutes

• Visualization and counterstaining:

  • DAB chromogen: 5-10 minutes (monitor under microscope)

  • Hematoxylin counterstain: 1-2 minutes

  • Dehydrate, clear, and mount

How can I validate the specificity of my EIF5A antibody?

Validating specificity is crucial for reliable results:

• Positive controls:

  • Use cell lines with confirmed EIF5A expression: HeLa, NIH/3T3, PC-3, SH-SY5Y

  • Include tissues known to express EIF5A: mouse brain, mouse testis

• Negative controls:

  • Omit primary antibody while performing all other steps

  • Use isotype control antibodies

  • Include knockdown samples if available

• Western blot validation:

  • Verify single band at expected molecular weight (18 kDa)

  • Confirm band disappearance in knockdown samples

• Cross-reactivity testing:

  • The antibody should be tested against human, mouse, and rat samples as these species reactivity have been validated

What common technical issues might arise with biotin-conjugated antibodies and how can I address them?

For EIF5A specifically, antigen retrieval is critical - always use the recommended TE buffer pH 9.0 for optimal results .

How can I use EIF5A antibodies to study ribosome stalling at specific sequence motifs?

EIF5A is particularly important for resolving ribosome pausing at challenging sequences. To investigate this function:

• Sequence-specific translation analysis:

  • Create reporter constructs containing known stalling sequences (Pro-Pro dipeptides, Pro-Gly-Gly, etc.)

  • Express in cells with and without EIF5A manipulation

  • Quantify translation efficiency of each reporter

  • Use biotin-conjugated EIF5A antibody to confirm knockdown efficiency

• High-resolution ribosome profiling:

  • Profile ribosomes in control and EIF5A-depleted cells

  • Analyze ribosome occupancy at specific tripeptide motifs

  • Research has shown that eIF5A depletion causes ~2-fold higher ribosome occupancy at polyproline stretches compared to control cells

• In vitro biochemical assays:

  • Set up reconstituted translation systems

  • Program with mRNAs containing stalling sequences

  • Measure peptide bond formation rates with and without eIF5A

  • Studies have demonstrated that eIF5A increases peptidyl-tRNA hydrolysis more than 17-fold during termination

How can I distinguish between hypusinated and non-hypusinated forms of EIF5A?

The hypusine modification is essential for EIF5A function. To differentiate between forms:

• 2D gel electrophoresis:

  • Separate proteins based on both pI and molecular weight

  • Western blot with biotin-conjugated EIF5A antibody

  • The hypusinated form will have a different pI than non-hypusinated EIF5A

• Specialized antibodies:

  • Use hypusine-specific antibodies in parallel with pan-EIF5A antibodies

  • Compare signal ratios to determine modification status

  • Perform comparative immunostaining to assess localization differences

• Mass spectrometry analysis:

  • Immunoprecipitate EIF5A using biotin-conjugated antibodies

  • Analyze by mass spectrometry to identify and quantify hypusinated vs. non-hypusinated peptides

  • This approach provides precise quantification of modification status

How can I study the dynamics of EIF5A's interaction with the ribosome?

To investigate EIF5A-ribosome interactions:

• Co-immunoprecipitation studies:

  • Use biotin-conjugated EIF5A antibodies to pull down EIF5A complexes

  • Analyze associated ribosomal proteins by Western blot

  • Compare interactions under different cellular conditions

• Proximity labeling approaches:

  • Express EIF5A fused to a proximity labeling enzyme

  • Activate labeling to tag proteins in close proximity

  • Purify biotinylated proteins and identify by mass spectrometry

• Real-time translation monitoring:

  • Set up single-molecule translation systems

  • Monitor ribosome movement with and without EIF5A

  • Analyze pause kinetics at specific sequence contexts

  • Correlate with ribosome profiling data showing accumulation of ribosomes at specific motifs

What approaches can I use to study EIF5A in disease models?

EIF5A has been implicated in various disease processes. Advanced approaches include:

• Patient-derived samples analysis:

  • Analyze EIF5A expression and modification in disease tissues

  • Use biotin-conjugated EIF5A antibodies with streptavidin-fluorophore detection

  • Perform co-localization studies with disease markers

  • Compare hypusination status between healthy and diseased samples

• Therapeutic targeting studies:

  • Use EIF5A antibodies to monitor effects of hypusination inhibitors

  • Validate target engagement in drug discovery pipelines

  • Assess changes in EIF5A-dependent translation

• Multi-omics integration:

  • Combine EIF5A immunoprecipitation with RNA-seq and proteomics

  • Identify disease-specific EIF5A-regulated mRNAs

  • Correlate with alterations in protein synthesis

How should I interpret changes in EIF5A localization under different cellular conditions?

EIF5A localization can provide insights into its function:

• Normal conditions:

  • EIF5A is predominantly cytoplasmic

  • Often shows enrichment in areas of active translation

  • May associate with specific cytoplasmic structures

• Stress conditions:

  • Potential relocalization to stress granules

  • Changes in nuclear-cytoplasmic distribution

  • Altered association with polysomes

• Quantitative analysis:

  • Use digital image analysis to quantify distribution patterns

  • Calculate nuclear/cytoplasmic ratios

  • Measure co-localization with markers of translation machinery

When interpreting these changes, consider that altered localization may reflect functional adaptations in translation regulation under different cellular states.

What can polysome profile changes tell me about EIF5A function?

Polysome profiles provide valuable functional insights:

How do I interpret ribosome profiling data in the context of EIF5A function?

Ribosome profiling provides genome-wide insights into translation:

• Key features to analyze:

  • Ribosome distribution along mRNAs (5' to 3' analysis)

  • Metagene analysis around start and stop codons

  • Enrichment of ribosomes at specific sequence motifs

• Typical findings in EIF5A-depleted cells:

  • Global elongation defect with ribosomes stalling at many sequences

  • ~2-fold higher ribosome occupancy at polyproline stretches

  • Accumulation of ribosomes at stop codons and in 3' UTRs, indicating termination defects

  • Ribosome queuing upstream of stalling sites

• Advanced analysis:

  • Calculate pause scores at different tripeptide motifs

  • Compare pause site sequences to identify common features

  • Correlate ribosome stalling with protein output

Understanding these patterns can reveal the sequence contexts where EIF5A is most critical for efficient translation.

How can I investigate non-canonical functions of EIF5A beyond translation?

Recent research suggests EIF5A may have functions beyond translation:

• RNA-binding studies:

  • Perform CLIP-seq using biotin-conjugated EIF5A antibodies

  • Identify direct RNA interactions

  • Analyze binding motifs and target RNAs

• Protein-protein interaction networks:

  • Use proximity labeling approaches

  • Perform co-immunoprecipitation with biotin-conjugated antibodies

  • Identify non-ribosomal interaction partners

• Subcellular localization studies:

  • Examine EIF5A localization to non-canonical sites

  • Perform high-resolution microscopy with biotin-conjugated antibodies and streptavidin-fluorophores

  • Analyze dynamic changes under different cellular conditions

What novel technologies can enhance EIF5A research?

Emerging technologies are expanding EIF5A research possibilities:

• CRISPR-based approaches:

  • Create endogenously tagged EIF5A to monitor native protein

  • Generate conditional knockout systems for temporal control

  • Establish hypusination-deficient mutants

• Real-time translation monitoring:

  • Apply single-molecule techniques to observe EIF5A function

  • Use ribosome profiling with short timepoints to capture dynamics

  • Develop biosensors for EIF5A activity

• Cryo-EM structural studies:

  • Determine high-resolution structures of EIF5A-ribosome complexes

  • Analyze conformational changes during elongation and termination

  • Use antibody fragments as structural probes

These emerging approaches will provide new insights into EIF5A's essential role in translation regulation and potential non-canonical functions.