EIF5 Antibody

What is EIF5 and why is it important in translation research?

EIF5 is a critical component of the 43S pre-initiation complex (43S PIC) that binds to the mRNA cap-proximal region, scans mRNA 5'-untranslated region, and locates the initiation codon. It functions as a GTPase-activating protein (GAP) by promoting GTP hydrolysis by eIF2G (EIF2S3). EIF5 interacts with both EIF1 (via its C-terminal domain) and EIF1A (via its N-terminal domain) during scanning, helping maintain EIF1 within the open 43S PIC. When a start codon is recognized, EIF5 induces eIF2G to hydrolyze GTP and stabilizes the PIC in its closed conformation .

The importance of EIF5 lies in its dual function: regulating P₁ release and stabilizing the closed PIC conformation, both of which contribute to stringent AUG selection in vivo. Mutations in EIF5 can alter these functions, affecting translation initiation accuracy and potentially leading to scanning past uORF1, which impacts translation derepression .

How do I determine the optimal EIF5 antibody dilution for my experiment?

Determining optimal antibody dilution requires systematic testing rather than relying solely on manufacturer recommendations. Based on published data, start with these application-specific ranges:

For optimization, prepare a dilution series and test on your specific sample types. The optimal dilution should provide strong specific signal with minimal background. For tissue samples, antigen retrieval methods significantly impact results - both citrate buffer (pH 6.0) and TE buffer (pH 9.0) have been effectively used with EIF5 antibodies, but comparative testing is recommended for your specific tissue .

Which fixation protocol is most suitable for EIF5 immunolocalization studies?

Fixation protocol selection critically impacts EIF5 immunolocalization results. Different fixation methods reveal distinct aspects of EIF5 subcellular distribution:

In comparative studies, EIF5 showed different localization patterns depending on fixation method. For comprehensive analysis, employ both methods in parallel experiments. Additionally, validation through GFP-EIF5 fusion protein expression has confirmed that these fixation-dependent localization patterns are genuine rather than artifacts .

How can I effectively use EIF5 antibodies to study translation initiation complex assembly?

To study translation initiation complex assembly using EIF5 antibodies, employ a multi-method approach:

• Co-immunoprecipitation (Co-IP) of initiation complexes:

  • Use anti-EIF5 antibodies (such as mouse monoclonal IgG2b or rabbit polyclonal alternatives) for immunoprecipitation

  • Analyze precipitates for associated factors (eIF1, eIF2, eIF3, eIF4G)

  • Include both non-denaturing and crosslinking protocols to capture transient interactions

• Sequential Co-IP approach:

  • First IP with anti-HA-eIF3 followed by Western blotting with anti-EIF5

  • Alternative: First IP with FLAG-eIF2 followed by probing for EIF5

  • This approach has successfully demonstrated that mutations in EIF5's basic area II substantially reduced HA-eIF3 binding to EIF5 and eliminated HA-eIF3 binding to eIF2α

• Gradient fractionation:

  • Fractionate lysates on sucrose gradients

  • Collect fractions and analyze for EIF5 by Western blotting

  • EIF5-containing complexes typically elute at approximately 360 mM KCl when using ion-exchange chromatography

These approaches have been instrumental in demonstrating that the eIF5 HEAT domain acts as a critical nucleation core for preinitiation complex assembly, with distinct surface areas mediating specific factor interactions .

What are the validated approaches for studying EIF5 mutations and their effects on start codon selection fidelity?

Studying EIF5 mutations requires sophisticated methodologies to assess their impact on start codon selection fidelity:

• Plasmid construction and mutagenesis approach:

  • Modify plasmids (like pAS5-101) by replacing unique restriction sites (e.g., NdeI site CATATG with AAGATG)

  • Construct TIF5 mutant alleles by fusion PCR

  • Insert PCR products between appropriate restriction sites (EcoRI and SalI) in vectors like YCplac111 (sc) or YEplac181 (hc)

  • Confirm all constructs by DNA sequencing

• In vivo reporter systems:

  • Utilize GCN4-lacZ reporters to monitor translation reinitiation efficiency

  • Employ dual luciferase reporters with near-cognate (UUG) and cognate (AUG) start codons to measure initiation fidelity

  • Mutations like G31R in eIF5 alter regulation of Pi release, accelerating it at UUG while decreasing it at AUG codons, resulting in increased UUG initiation

• Suppressor analysis:

  • Test suppressor mutations (like G62S and M18V) in the eIF5 context

  • Assess their ability to mitigate defects caused by primary mutations

  • For example, suppressor G62S mitigates both defects of G31R, while suppressor M18V impairs GTP hydrolysis with little effect on PIC conformation

This methodological framework has revealed that both eIF5's functions—regulating Pi release and stabilizing the closed PIC conformation—contribute to stringent AUG selection in vivo .

How can I distinguish between EIF5 and EIF5A in my experimental system?

Distinguishing between EIF5 and EIF5A, which perform distinct functions in translation, requires careful experimental design:

• Antibody selection criteria:

  • Use antibodies raised against non-homologous regions

  • Select EIF5 antibodies targeting epitopes near C-terminus (residues near carboxy terminus or aa 100-150 )

  • EIF5A-specific antibodies should target unique features like the hypusine modification site

• Molecular weight differentiation:

  • EIF5: ~49-58 kDa (observed molecular weight often ~50 kDa )

  • EIF5A: ~17-20 kDa

  • Use appropriate gel concentration (10-12% for resolving both)

• Functional assessment:

  • EIF5 primarily functions in initiation

  • EIF5A functions in elongation, particularly with polyproline motifs, and termination

  • Design knockdown experiments targeting each factor separately and assess polysome profiles (after 10 hr of EIF5A depletion, an increase in polysome/monosome ratio is observed )

• Subcellular localization:

  • EIF5A shows distinct nuclear-cytoplasmic shuttling (interacts with CRM1)

  • EIF5 predominantly localizes with translation initiation machinery

  • Use double immunofluorescence with markers for specific compartments (e.g., calnexin for ER, CRM1 for nuclear transport)

How should I address non-specific binding when using EIF5 antibodies in immunoprecipitation?

Non-specific binding in EIF5 immunoprecipitation experiments can be systematically minimized through the following protocol optimizations:

• Pre-clearing strategy:

  • Pre-clear lysates with appropriate control IgG and protein A/G beads for 1 hour at 4°C

  • Use at least 20 μl beads per 1 mg of total protein

  • Pre-blocking beads with BSA (1%) and tRNA (0.1 mg/ml) further reduces background

• Buffer optimization:

  • Titrate salt concentration (100-500 mM KCl) - EIF5 typically elutes at ~360 mM KCl

  • Include detergents at optimal concentrations: 0.1-0.5% NP-40 or 0.1% Triton X-100

  • Add competitors for non-specific interactions: 0.1-0.5% BSA, 0.1-0.5 mg/ml tRNA

• Antibody selection and validation:

  • Use antibodies with validated IP applications (e.g., for human samples, antibody clone EPR12140(B) has demonstrated specificity )

  • Consider using epitope-tagged EIF5 constructs (FLAG-tagged EIF5 constructs have been successfully employed )

  • Use antibody quantities proportional to lysate concentration (typically 0.5-4.0 μg antibody for 1.0-3.0 mg total protein)

• Washing protocol optimization:

  • Implement graduated washing with decreasing detergent concentrations

  • Perform at least 4-5 washes with buffer volumes 10× the bead volume

  • Short wash incubations (1 min) with gentle rocking rather than vortexing

These optimizations have proven effective in studies investigating EIF5's interactions with other translation factors, particularly in characterizing the binding domains within the eIF5 HEAT domain .

What controls are essential when using EIF5 antibodies for subcellular localization studies?

For rigorous subcellular localization studies with EIF5 antibodies, implement these essential controls:

• Antibody specificity controls:

  • Pre-absorption control: Incubate antibody with recombinant EIF5 protein before staining (should eliminate specific signal)

  • Peptide competition: Use the immunizing peptide to block specific binding

  • Secondary antibody only: Verify lack of non-specific binding

• Expression construct validation:

  • Compare GFP-EIF5 fusion protein localization with antibody staining patterns

  • Validate fusion protein expression by Western blot

  • Compare direct visualization of GFP-EIF5 with immunofluorescent staining of the same cells using anti-EIF5 antibodies

• Fixation method controls:

  • Compare paraformaldehyde (4%) with methanol fixation

  • Document localization differences between methods

  • Use orthogonal methods to confirm patterns observed with each fixation

• Co-localization markers:

  • Include markers for specific cellular compartments (ER: calnexin; nuclear transport: CRM1)

  • Quantify co-localization using appropriate statistical methods

  • Account for cell cycle stage which may affect EIF5 distribution

These controls enabled researchers to establish that EIF5 shows different distribution patterns depending on fixation method, with partial co-localization with calnexin (ER marker) in formaldehyde-fixed cells and interactions with nuclear transport machinery (CRM1) .

How can I optimize Western blotting protocols specifically for EIF5 detection?

Optimizing Western blotting for EIF5 detection requires addressing specific challenges related to this protein:

• Sample preparation refinements:

  • Use phosphatase inhibitors (sodium fluoride, sodium orthovanadate) and protease inhibitors

  • Optimal lysis buffer: 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 1% Triton X-100

  • Brief sonication (3 × 5 sec pulses) improves EIF5 extraction

• Gel and transfer parameters:

  • Use 10% SDS-PAGE for optimal resolution of EIF5 (MW ~49-58 kDa)

  • Transfer conditions: 100V for 60 minutes using PVDF membrane (0.45 μm)

  • Wet transfer yields better results than semi-dry for EIF5

• Detection optimization:

  • Primary antibody incubation: 1:1000 dilution, overnight at 4°C in 5% BSA (preferred over milk for phospho-specific detection)

  • Secondary antibody: HRP-conjugated anti-rabbit/mouse IgG at 1:5000-1:10000

  • Signal development: Enhanced chemiluminescence with 1-5 minute exposure

• Validated antibody performance data:

These optimizations are based on published protocols that successfully detected endogenous EIF5 in multiple experimental systems .

How can EIF5 antibodies be applied to study stress granule dynamics during cellular stress?

EIF5 antibodies offer valuable tools for investigating stress granule (SG) dynamics during cellular stress responses:

• Co-localization analysis protocol:

  • Induce stress granules with arsenite (0.5 mM, 30 min), thapsigargin, or heat shock

  • Co-stain for EIF5 and established SG markers (G3BP1, TIA-1, PABP)

  • Quantify co-localization using Pearson's or Manders' coefficients

  • EIF5's recruitment to SGs provides insights into translational regulation during stress

• Time-course dynamics:

  • Perform time-resolved immunofluorescence after stress induction

  • Monitor EIF5 redistribution at 0, 15, 30, 60, and 120 minutes

  • Correlate with polysome profile changes to link EIF5 localization with translation status

• Proximity ligation assay (PLA) methodology:

  • Use EIF5 antibodies in combination with antibodies against stress granule components

  • PLA signal indicates proximity (<40 nm) between EIF5 and target proteins

  • This approach has revealed novel interactions between translation factors in stress conditions

• Functional assessment through mutation analysis:

  • Express wild-type vs. mutated EIF5 (e.g., G31R, which affects GTP hydrolysis)

  • Compare stress granule recruitment patterns

  • Evaluate translational recovery kinetics after stress relief

This approach has advanced our understanding of how translation initiation factors contribute to stress response mechanisms through dynamic subcellular redistribution and functional reorganization.

What methodological approaches can distinguish between the dual functions of EIF5 in translation initiation?

Distinguishing between EIF5's functions as a GTPase-activating protein (GAP) and a stabilizer of the closed pre-initiation complex requires sophisticated experimental design:

• Domain-specific mutation analysis:

  • N-terminal domain (NTD) mutations primarily affect GAP activity

  • C-terminal domain (CTD) mutations primarily affect stabilization function

  • Specific mutations like G31R alter regulation of Pi release, accelerating it at UUG while decreasing it at AUG codons

  • Suppressor mutations like G62S mitigate both defects of G31R, while M18V impairs GTP hydrolysis with little effect on PIC conformation

• Reconstituted in vitro translation system:

  • Assemble 43S complexes with purified components

  • Add radiolabeled GTP to monitor hydrolysis

  • Use non-hydrolyzable GTP analogs to separate binding from catalysis

  • Measure Pi release kinetics at AUG vs. UUG codons

• Structural analysis approach:

  • Cryo-EM analysis of 48S PICs with EIF5-NTD

  • Multiple maps (Maps A, B, C1, C2) showing clear densities for EIF1A, EIF3, TC, EIF5, and mRNA

  • Analysis reveals that β-hairpin 1 of eIF5-NTD monitors codon:anticodon interaction similar to eIF1

  • Key residues (Lys24, Gly27, Arg28, Gly29, Asn30, Gly31, Lys71, Arg73) make extensive contacts with tRNA

• In vivo reporter assays:

  • Use reporters with near-cognate (UUG) and cognate (AUG) start codons

  • Quantify GTP hydrolysis and Pi release rates for each codon type

  • Evaluate effects of mutations on the partitioning of PICs between open and closed states

These approaches have revealed that EIF5's dual functions contribute differentially to translation initiation fidelity, with both mechanisms collaborating to ensure stringent AUG selection in vivo .

How can researchers assess the role of EIF5 in translation regulation during disease states?

Investigating EIF5's role in disease states requires integrating multiple research approaches:

• Tissue expression profiling protocol:

  • Compare EIF5 expression levels across normal vs. diseased tissues

  • Use validated antibodies (like EPR12140(B)) for immunohistochemistry

  • Optimize antigen retrieval: TE buffer pH 9.0 or citrate buffer pH 6.0

  • Quantify expression using H-score or digital analysis software

• Polysome profiling methodology:

  • Fractionate ribosomes from disease vs. control samples

  • Analyze EIF5 distribution across fractions by Western blotting

  • Altered polysome/monosome ratios may indicate translation dysregulation

  • EIF5 depletion typically increases polysome/monosome ratio

• Phosphorylation state analysis:

  • Use phospho-specific antibodies or phospho-proteomic approaches

  • Monitor EIF5 phosphorylation status in response to disease signals

  • Correlate with changes in translation efficiency of specific mRNAs

• Therapeutic targeting assessment:

  • Design competitive peptides mimicking EIF5-binding interfaces

  • Test compounds that selectively modulate EIF5's GAP activity

  • Evaluate effects on translation of disease-relevant mRNAs

  • Monitor cell viability, proliferation, and disease phenotypes

This integrated approach has proven valuable in understanding how alterations in translation initiation contribute to disease pathogenesis and in identifying potential therapeutic targets within the translation machinery.