EIF5B Antibody

What is EIF5B and what is its primary function in cellular processes?

EIF5B (also known as IF2, KIAA0741) is a eukaryotic translation initiation factor that plays a significant mechanical role in the initiation of mRNA translation. It functions as a ribosome-dependent GTPase that promotes the joining of the 60S ribosomal subunit to the pre-initiation complex to form the 80S initiation complex with the initiator methionine-tRNA in the P-site base paired to the start codon .

Together with eIF1A (EIF1AX), EIF5B actively orients the initiator methionine-tRNA in a conformation that allows 60S ribosomal subunit joining . Its GTPase activity is not essential for ribosomal subunits joining, but GTP hydrolysis is needed for eIF1A ejection quickly followed by EIF5B release to form elongation-competent ribosomes .

What is the molecular structure and weight of EIF5B?

EIF5B is a multidomain protein with a calculated molecular weight of approximately 139 kDa, though it is sometimes observed at around 175 kDa on Western blots . The protein consists of multiple functional domains with specific roles:

The protein structure includes an intrinsically disordered N-terminal region that has been found to stimulate IRES usage .

What are the common applications for EIF5B antibodies in research?

EIF5B antibodies are used in multiple research applications:

These antibodies have been validated with human, mouse, and rat samples, with some commercial antibodies also predicted to work with pig, horse, sheep, rabbit, dog, chicken and Xenopus samples .

How should I optimize EIF5B antibody dilutions for different experimental applications?

Optimization depends on your specific application and sample type:

For Western Blot:

• Begin with a 1:1000 dilution and adjust as needed

• For mouse brain tissue and A549 cells, 1:500-1:2000 has shown good results

• Include positive controls such as human A549 cells or mouse brain tissue

For Immunohistochemistry:

• Start with 1:100 dilution

• Antigen retrieval with TE buffer pH 9.0 is suggested

• Alternatively, citrate buffer pH 6.0 can be used

For Immunofluorescence:

• Start with 1:50-1:100 dilution

• MCF-7 cells have been successfully used for validation

It is recommended that each antibody should be titrated in your specific testing system to obtain optimal results. Sample-dependent variations may require adjustments to standard protocols .

What controls should I include when performing experiments with EIF5B antibodies?

For rigorous experimental design, include:

• Positive controls:

  • Cell lines known to express EIF5B (A549, MCF-7 cells)

  • Mouse brain tissue

• Negative controls:

  • EIF5B knockdown/knockout samples using siRNA or CRISPR-Cas9 approaches

  • Multiple studies have used the FKBP-dTag system for specific depletion of EIF5B

• Loading controls:

  • For Western blots: housekeeping proteins like β-actin

  • For immunohistochemistry: serial sections with secondary antibody only

How can I validate the specificity of an EIF5B antibody?

Several approaches for antibody validation:

• siRNA knockdown: Compare antibody signal in control vs. EIF5B-depleted cells

  • Research has shown ~90% reduction in EIF5B protein levels can be achieved using a pool of three EIF5B-specific siRNAs

• CRISPR-Cas9 knockout or knockdown:

  • FKBP-dTag system can be used for rapid depletion of EIF5B

  • This system allows depletion of EIF5B in less than 30 minutes

• Immunoblotting with recombinant protein:

  • Use purified EIF5B protein as a positive control

  • Test antibody recognition of specific domains using truncated constructs

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Signal should be significantly reduced or eliminated

What experimental approaches can be used to study EIF5B's role in translation initiation?

Several methodologies have been established:

• In vitro translation assays:

  • Rabbit reticulocyte lysate (RRL) supplemented with purified EIF5B

  • Program with firefly luciferase reporter mRNAs synthesized, capped, and polyadenylated in vitro

• Ribosome binding assays:

  • Protocol: Incubate 30 pmol EIF5B with 15 pmol ribosomes and 0.1 mM GDPNP

  • Centrifuge through sucrose cushion to isolate ribosome-bound fractions

  • Analyze by SDS-PAGE and quantify using image analysis software

• Cryo-EM structural studies:

  • Assembly of late initiation complex with EIF5B stalled at the 80S ribosome

  • Preincubation of 40S with initiation factors eIF1 and eIF1A and mRNA

  • Addition of Met-tRNAiMet as a TC with eIF2 and GTP

• GTPase activity assays:

  • Monitor GTP hydrolysis using radioactive or fluorescent GTP analogs

  • Assess impact of mutations on GTPase activity

How does EIF5B's domain structure relate to its functional mechanism?

EIF5B's multidomain architecture enables it to integrate multiple inputs:

• Domain-specific functions:

  • Domain IV: Projects into intersubunit space to recognize properly delivered Met-tRNAiMet; also interacts with other factors like eIF1A and eIF5

  • Domain III: Couples the rotational state of the 40S to the G-domain

  • Domain II: Provides additional anchoring points to the 40S

  • G-domain: Responsible for GTP binding and majority of interactions with the large subunit

• Key structural elements:

  • Tyrosine 837 and its flanking residues are critical for coordinating different inputs needed for progression into elongation

  • These residues are key in interdomain communications in EIF5B for proper discrimination of ribosomal states

• Conformational changes:

  • Long-range interdomain communications regulate GTP hydrolysis

  • CD spectrometry can be used to assess structural integrity of EIF5B variants

What is the difference between EIF5B's role in canonical vs. IRES-mediated translation?

EIF5B has distinct roles in different translation pathways:

The intrinsically disordered N-terminal region of EIF5B preferentially promotes IRES activity through a non-canonical mechanism . In vitro translation assays in rabbit reticulocyte lysate have confirmed that addition of EIF5B directly stimulates translation of IRES-containing transcripts over cap-dependent reporters .

How does EIF5B interact with other translation initiation factors?

EIF5B functionally interacts with several factors:

• eIF1A:

  • EIF5B and eIF1A together orient the initiator methionine-tRNA

  • The last five residues of eIF1A are critical for EIF5B binding

  • Substituting Ala for these residues (eIF1A-5A) impairs EIF5B binding

• eIF5:

  • Together with eIF5, EIF5B stimulates 48S initiation complex formation

  • Both factors have been shown to work together on mRNAs with non-optimal initiation context

• Release mechanism:

  • GTP hydrolysis is needed for eIF1A ejection and EIF5B release

  • In the presence of nonhydrolyzable GDPNP, both factors remain associated with 80S complexes

  • With GTP, these factors are released

• Experimental approaches:

  • Immunoblot analysis using antibodies against eIF5B, eIF1A, eIF3b, eIF5, eIF2γ, eIF2α, and RPS22

  • Co-immunoprecipitation to detect factor interactions

  • Overexpression studies to test functional relationships

How can I design experiments to study EIF5B's role in cancer cell survival?

EIF5B has been implicated in cancer cell survival, particularly in glioblastoma multiforme (GBM):

• siRNA-mediated depletion studies:

  • siRNA-mediated depletion of EIF5B increases sensitivity of GBM cells to treatments

  • Effect is specific to cancer cells, not observed in immortalized fibroblasts

  • Protocol: Use a pool of three EIF5B-specific siRNAs to achieve ~90% reduction in protein levels

• Cell viability assays:

  • alamarBlue assay can be used to measure cell viability

  • Compare different cell lines with diverse genetic backgrounds (p53, PTEN, EGFR, and MGMT status)

• Experimental design considerations:

  • Test in multiple GBM cell lines (U343, U251N, A172, U373, U87MG)

  • Include non-cancerous control cell lines (HEK293T, WI-38 lung fibroblasts)

  • Consider genetic background variations that might influence results

• Mechanistic studies:

  • Investigate EIF5B's role in stress response pathways

  • Examine how EIF5B might substitute for eIF2 under stress conditions

  • Study translation of specific mRNAs important for cancer cell survival

What are common issues when using EIF5B antibodies and how can they be resolved?

For optimal results with EIF5B antibodies, prepare samples fresh with protease inhibitors and optimize lysis conditions for this high molecular weight protein. Store antibodies according to manufacturer recommendations—typically at -20°C in aliquots .

How can I study the GTPase activity of EIF5B and its mutants?

To investigate EIF5B's GTPase function:

• GTPase-deficient mutants:

  • eIF5B-T439A mutation has been shown to accumulate on 80S complexes in vivo

  • This mutant was retained along with eIF1A on 80S complexes formed in vitro

• In vitro GTPase assays:

  • Use purified EIF5B protein (wild-type and mutants)

  • Measure GTP hydrolysis using [γ-32P]GTP or fluorescent GTP analogs

  • Compare activity with different nucleotide analogs (GTP, GDP, GDPNP)

• Structural considerations:

  • Tyrosine 837 is key in coordinating inputs needed for GTP hydrolysis

  • Long-range interdomain communications regulate GTPase activity

  • Consider testing mutations in different domains to assess their impact

• Ribosome-binding assays:

  • Compare binding of wild-type and GTPase-deficient mutants to ribosomes

  • Assess effects of GTP vs. non-hydrolyzable analogs (GDPNP)

These approaches can provide insights into how EIF5B's GTPase activity is regulated and its role in translation initiation.

What is known about EIF5B's role under cellular stress conditions?

Under stress conditions, EIF5B can substitute for eIF2:

• When eIF2 is phosphorylated (preventing Met-tRNAiMet delivery), EIF5B can:

  • Operate the eukaryotic initiation machinery in a "bacterial-like" mode

  • Substitute for eIF2 in delivering Met-tRNAiMet to the P site

• AUG selection accuracy:

  • EIF5B plays a regulatory role in preventing identification of correct AUG codons

  • Forces scanning machinery to assemble initiation complexes at upstream AUGs (uAUGs)

  • Works with eIF5, eIF1A, and the 40S subunit in this process

• Research approaches:

  • Induce cellular stress (e.g., with thapsigargin or arsenite)

  • Monitor eIF2 phosphorylation and EIF5B activity

  • Use reporter constructs with uAUGs to study AUG selection accuracy

What new methodologies are being developed to study EIF5B function?

Emerging techniques for EIF5B research:

• Rapid protein depletion systems:

  • FKBP-dTag system allows depletion of EIF5B in less than 30 minutes

  • Uses dTagV-1 small molecule to target FKBP-tagged EIF5B for degradation

  • Procedure: Use CRISPR-Cas9 to edit endogenous eIF5B, fusing HA tags and FKBPV12 degron to N-terminus

• Single-molecule approaches:

  • Real-time, single-molecule experiments reveal dynamics of eukaryotic initiation

  • Long residence time observed for EIF5B on 80S complex after 60S recruitment

  • Rearrangement of 80S-IC during EIF5B residence is proposed

• Cryo-EM structural studies:

  • High-resolution structures of EIF5B bound to ribosomes

  • Visualization of specific interactions and conformational changes

  • Identification of key residues in interdomain communications

These advanced techniques are providing new insights into EIF5B's complex role in translation initiation and regulation.