EIF4G3 Antibody, Biotin conjugated

What is EIF4G3 and what cellular functions does it perform?

EIF4G3 functions as an essential scaffold protein within the eukaryotic translation initiation 4F (EIF4F) complex. This complex mediates the rate-limiting initial step of translation by assembling on the 7-methylguanosine cap structure of mRNAs. EIF4G3 facilitates the formation of a closed-loop mRNA structure by interacting with poly(A)-binding protein (PABP) as well as with the 5' cap, contributing to the unwinding of secondary structures in the 5' untranslated region and recruitment of the 40S ribosomal subunit and additional translation factors . Beyond translation initiation, EIF4G3 has been implicated in nuclear mRNA biogenesis and surveillance processes, suggesting multifunctional roles in RNA metabolism . The protein is particularly notable for its crucial role in spermatogenesis, as demonstrated by studies showing that mutations in the Eif4g3 gene lead to male infertility in mice .

How does EIF4G3 interact with other components of the translation machinery?

EIF4G3 serves as a critical interaction hub within the EIF4F complex, which includes the cap-binding protein EIF4E and the ATP-dependent RNA helicase EIF4A. The hypophosphorylated form of translation repressors like EIF4EBP3 competes with EIF4G1/EIF4G3 for binding to EIF4E, thereby inhibiting translation. When EIF4EBP3 becomes hyperphosphorylated, it dissociates from EIF4E, allowing EIF4G1/EIF4G3 to interact with EIF4E and initiate translation . This regulatory mechanism highlights how EIF4G3 functions as a critical control point for translation initiation. The interactions between EIF4G3 and other translation components are frequently studied using specific antibodies to detect binding partners and characterize complex formation under various cellular conditions .

What is the subcellular localization pattern of EIF4G3 in different cell types?

Surprisingly, EIF4G3 demonstrates unexpected subcellular localization patterns that challenge traditional views of translation factors. While conventional understanding would predict cytoplasmic localization for translation factors, immunofluorescence studies have revealed that EIF4G3 is prominently located in the nucleus of spermatocytes. Even more surprisingly, it is highly enriched in the XY body, the chromatin domain formed by transcriptionally inactive sex chromosomes . This unexpected nuclear localization pattern suggests additional functions for EIF4G3 beyond its canonical role in cytoplasmic translation. The localization varies across cell types and developmental stages, with particularly notable expression patterns in male germ cells. This distinctive localization pattern can be effectively visualized using specific antibodies against EIF4G3 in immunofluorescence experiments .

What are the key technical specifications of commercially available EIF4G3 antibodies?

EIF4G3 antibodies are available with various specifications to suit different experimental needs. Polyclonal antibodies raised in rabbits are commonly used for EIF4G3 detection, with some products specifically recognizing epitopes between amino acids 222-271 of human EIF4G3 . These antibodies typically demonstrate reactivity across multiple species including human, mouse, rat, cow, and monkey, with predicted reactivity extending to additional species based on sequence homology . For optimal results in various applications, researchers should select antibodies validated for their specific needs. For instance, some antibodies are specifically validated for Western blotting (WB) applications while others may be optimized for immunohistochemistry (IHC) or immunofluorescence (IF) . Available products include unconjugated antibodies as well as those conjugated to biotin, FITC, or HRP for specialized detection methods .

What advantages does biotin conjugation offer when using EIF4G3 antibodies?

Biotin conjugation provides several methodological advantages for EIF4G3 detection in research applications. The strong binding affinity between biotin and streptavidin/avidin creates an amplification system that can significantly enhance detection sensitivity compared to unconjugated primary antibodies. This feature is particularly valuable when studying proteins with low expression levels or when examining subtle changes in protein localization or quantity. Biotin-conjugated antibodies offer flexible detection options, allowing researchers to use various streptavidin-conjugated detection reagents (HRP, fluorophores, gold particles) without needing to change the primary antibody system. This adaptability makes biotin-conjugated EIF4G3 antibodies suitable for multiple experimental platforms, including ELISA, immunocytochemistry, flow cytometry, and imaging applications . Additionally, the biotin-streptavidin system generally produces lower background signals compared to direct enzyme conjugation methods.

How should dilution factors be optimized when using EIF4G3 antibodies?

Determining optimal dilution factors for EIF4G3 antibodies requires systematic optimization based on the specific application and antibody properties. Published research using EIF4G3 antibodies suggests starting dilutions of 1:100 for immunofluorescence and immunohistochemistry applications, and 1:3000 for Western blotting . For instance, studies examining EIF4G3 nuclear localization in spermatocytes successfully employed rabbit anti-EIF4G3 antibodies (Bethyl, A301-769A) at 1:100 dilution for immunofluorescence . Western blot applications typically use more dilute antibody concentrations, with successful detection reported using rabbit anti-EIF4G3 antibodies (Thermo, PA5-31101) at 1:3000 dilution . Researchers should perform titration experiments with their specific antibody lot, tissue type, and detection system to determine optimal concentrations that maximize specific signal while minimizing background. Consider factors such as fixation method, antigen retrieval technique, and incubation conditions when optimizing dilution protocols.

What protocols yield optimal results for detecting nuclear EIF4G3 in spermatocytes?

For optimal detection of nuclear EIF4G3 in spermatocytes, researchers should employ specialized protocols that preserve nuclear architecture while maintaining antigen accessibility. Begin with freshly isolated testicular tissue and prepare spermatocytes using either surface spreading or whole-mount preparation techniques. For whole-mount preparations, place spermatocytes on poly-L-lysine precoated slides for 30 minutes, fix with 4% paraformaldehyde (PFA) for 30 minutes, then permeabilize with 0.3% Triton X-100 for 15 minutes . Block with 5% bovine serum albumin for 30 minutes before applying EIF4G3 primary antibody (Bethyl, A301-769A) at 1:100 dilution overnight at 4°C . For visualization, use secondary antibodies conjugated with fluorophores such as Alexa Fluor 594 or 488 at 1:500 dilution . To specifically examine XY body localization, combine EIF4G3 antibody with markers for the XY body domain. High-resolution imaging is crucial - researchers have successfully used Zeiss Axio Imager Z2 or Leica SP5 confocal microscopes to capture the distinct nuclear localization pattern of EIF4G3 . This approach will reveal the characteristic enrichment of EIF4G3 in the XY body of spermatocytes.

How can researchers effectively validate the specificity of EIF4G3 antibodies?

Validating antibody specificity is crucial for reliable experimental outcomes, especially when studying proteins like EIF4G3 with homologous family members. A comprehensive validation approach should include multiple complementary techniques. Begin with knockout or knockdown controls where the target protein is depleted using gene editing (CRISPR/Cas9) or RNA interference approaches. For instance, researchers studying EIF4G3 have used Eif4g3-specific conditional knockout mice to validate antibody specificity . Peptide competition assays, where the antibody is pre-incubated with the immunizing peptide before application to samples, can confirm binding specificity. Cross-reactivity testing against related proteins (EIF4G1, EIF4G2) is essential, as these share structural domains with EIF4G3. Western blot analysis should demonstrate a single band of appropriate molecular weight, while immunoprecipitation followed by mass spectrometry can provide definitive confirmation of target specificity. Finally, comparing staining patterns across multiple antibodies targeting different epitopes of EIF4G3 can build confidence in observed localization patterns, particularly for the unexpected nuclear localization .

What experimental approaches can resolve the unexpected nuclear localization of EIF4G3?

The unexpected nuclear localization of EIF4G3, particularly its enrichment in the XY body of spermatocytes, requires specialized experimental approaches to fully characterize and validate this phenomenon. Researchers should employ subcellular fractionation techniques combined with Western blotting to biochemically confirm the presence of EIF4G3 in nuclear fractions. Super-resolution microscopy (STED, STORM, or SIM) can provide detailed visualization of the precise subnuclear localization beyond what conventional confocal microscopy offers. Co-immunoprecipitation studies from nuclear extracts can identify nuclear interaction partners that might explain this localization pattern. To establish functional relevance, researchers should design targeted mutations in potential nuclear localization signals within EIF4G3 and assess how these affect its localization and function in spermatogenesis. Live-cell imaging using fluorescently tagged EIF4G3 can track its dynamic localization during meiotic progression. Chromatin immunoprecipitation sequencing (ChIP-seq) would determine if EIF4G3 associates with specific genomic regions within the XY body. Combined immunofluorescence with RNA fluorescence in situ hybridization (RNA-FISH) could reveal associations between EIF4G3 and specific RNA transcripts in the nucleus .

How can translation activity be monitored in spermatocytes using puromycin labeling in conjunction with EIF4G3 localization?

Puromycin labeling provides a powerful approach for visualizing active translation in spermatocytes while simultaneously examining EIF4G3 localization. This technique relies on puromycin incorporation into nascent polypeptide chains, effectively tagging sites of active translation. Implement this approach by suspending germ cells in MEM alpha containing 5% fetal bovine serum, lactic acid, HEPES, and antibiotics in four-well plates and incubating for 1 hour . Then add 5 μM puromycin (Sigma P8833) along with 200 μM emetine (Sigma E2375) to prevent puromycin release from ribosomes, and incubate for 5 minutes . After brief centrifugation at 100g for 3 minutes and a PBS wash, prepare cells using surface spreading or whole-mount techniques . Perform dual immunofluorescence using anti-puromycin monoclonal antibody to detect translation sites and anti-EIF4G3 antibody to visualize EIF4G3 localization. This approach can reveal spatial relationships between EIF4G3-enriched domains (particularly the XY body) and active translation sites. The technique is particularly valuable for testing hypotheses about whether the XY body serves as a storage site for translation factors or plays a direct role in translational regulation during meiosis .

What is the significance of EIF4G3 mutations in male infertility and how can researchers investigate this connection?

The connection between EIF4G3 mutations and male infertility represents a significant research area with clinical implications. Studies in mice have demonstrated that mutation of the Eif4g3 gene causes male infertility with meiotic arrest at the end of prophase . To investigate this connection, researchers should employ a comprehensive experimental approach. Start by characterizing the specific meiotic defects using histological analysis of testicular sections from Eif4g3 mutant models. The repro8 mutation, a single base change in the last exon producing an ala-pro amino acid substitution, has been established as a valuable model . Analyze chromosome dynamics during meiosis using immunofluorescence staining for synaptonemal complex proteins and DNA damage markers. Assess translation of key meiotic transcripts using polysome profiling combined with RNA-seq and proteomics to identify which specific proteins fail to be properly translated in the absence of functional EIF4G3. Examine potential human infertility cases for EIF4G3 variants through targeted sequencing of infertile male cohorts. Create cellular models using patient-derived iPSCs differentiated toward the germ cell lineage to test the effect of specific mutations. As demonstrated in published research, developing conditional knockout models (such as Eif4g3 exon 5-specific conditional knockout mice) provides powerful tools for understanding stage-specific requirements for EIF4G3 during spermatogenesis .

How can the interactions between EIF4G3 and other translation factors in the XY body be comprehensively characterized?

Characterizing interactions between EIF4G3 and other translation factors in the XY body requires sophisticated approaches that integrate multiple techniques. Begin with proximity ligation assay (PLA) to visualize and quantify protein-protein interactions in situ, focusing on potential partners like EIF4E, EIF4A, and PABP within the XY body domain. Published research has already demonstrated that many translation-related proteins, though not all, co-localize with EIF4G3 in the XY body . Implement BioID or APEX2 proximity labeling by fusing these enzymes to EIF4G3, enabling the biotinylation of proximal proteins specifically within the XY body microenvironment. Isolated XY bodies can then be subjected to mass spectrometry to identify the complete interactome. Advanced imaging approaches such as Förster resonance energy transfer (FRET) or fluorescence lifetime imaging microscopy (FLIM) can detect direct protein interactions with nanometer resolution within intact nuclei. Complement these approaches with biochemical analyses including co-immunoprecipitation from purified XY body fractions followed by immunoblotting or mass spectrometry. ChIP-seq for EIF4G3 combined with RNA-seq can determine if these interactions relate to specific RNA transcripts or genomic regions. Finally, develop XY body-specific disruption of individual interactions through targeted mutations or peptide inhibitors to assess functional consequences on spermatocyte development and translation .

What are the optimal methods for quantifying EIF4G3 expression levels across different tissues or experimental conditions?

For precise quantification of EIF4G3 expression levels across different tissues or experimental conditions, researchers should employ complementary approaches at both RNA and protein levels. For RNA quantification, quantitative RT-PCR using gene-specific primers designed to span introns and amplify all known transcript variants provides reliable expression data. Primer pairs such as forward: 5'-GAT GCA GGA CAA AGC AGA GTC-3' and reverse: 5'-TAA CAC TTC ATC TGG GGT TCG-3' have been successfully used for Eif4g3 expression analysis . Results should be normalized to housekeeping genes such as Gapdh using the standard curve method . For protein quantification, Western blotting with carefully validated antibodies allows relative quantification between samples. Fluorescence-based Western detection systems offer superior linear dynamic range compared to chemiluminescence for more accurate quantification. For absolute quantification, consider using recombinant EIF4G3 protein standards of known concentration. Tissue microarrays or single-cell approaches can provide spatial information about expression patterns. The differential expression of EIF4G3 across cell types and developmental stages requires careful consideration of appropriate control samples. For example, when studying testicular expression, stage-specific isolation of germ cells allows precise tracking of expression changes during spermatogenesis. RNA-seq and proteomics approaches provide more comprehensive datasets but require sophisticated bioinformatic analysis pipelines for accurate quantification .

How do EIF4G3 expression patterns correlate with specific developmental stages in spermatogenesis?

The expression pattern of EIF4G3 shows specific correlations with developmental stages in spermatogenesis, providing insights into its functional role. Quantitative RT-PCR analysis demonstrates distinct temporal regulation of EIF4G3 during testicular development and throughout specific stages of spermatogenesis . The protein exhibits particularly notable expression during meiotic prophase, consistent with its critical function at this stage as evidenced by the meiotic arrest phenotype in Eif4g3 mutant mice . Importantly, immunofluorescence studies reveal dynamic changes in subcellular localization throughout spermatogenesis. EIF4G3 transitions from a predominantly cytoplasmic distribution in early spermatogenic cells to a striking nuclear localization pattern in spermatocytes, with specific enrichment in the XY body . This localization pattern suggests stage-specific functions beyond traditional translation initiation, potentially including roles in nuclear mRNA processing or preparation for post-meiotic translation. The expression and localization changes correlate with critical transitions in gene expression regulation during spermatogenesis, particularly the shift from transcriptional to translational control that occurs as spermatids develop and their nuclei condense. Researchers investigating these patterns should employ stage-specific isolation techniques combined with both RNA and protein analysis methods to fully characterize the dynamic expression profile throughout spermatogenesis .

How do the properties and functions of EIF4G3 compare with other EIF4G family members?

Table 1: Comparative characteristics of EIF4G family members based on immunofluorescence studies and genetic analyses .

What methodological approaches can distinguish between the functions of different EIF4G proteins in translation regulation?

Distinguishing between the functions of different EIF4G proteins in translation regulation requires sophisticated methodological approaches that can isolate their specific contributions. RNA interference or CRISPR-based approaches targeting individual EIF4G family members (EIF4G1, EIF4G2, EIF4G3) allow assessment of isoform-specific functions through comparative analysis of translation efficiency, polysome profiles, and cellular phenotypes. Rescue experiments using mutant constructs resistant to knockdown can confirm specificity and identify critical functional domains. Ribosome profiling combined with RNA sequencing following isoform-specific depletion can reveal which mRNA subsets depend on particular EIF4G proteins for efficient translation. For studying effects on specific pathways, reporter constructs containing various 5' UTR regulatory elements can determine if certain translation regulation mechanisms preferentially involve specific EIF4G isoforms. Proximity labeling approaches (BioID, APEX) with different EIF4G family members as baits can identify unique interaction partners that might explain their non-redundant functions. The development of isoform-specific inhibitors or domain-specific antibodies that can block particular interactions would provide powerful tools for acute functional perturbation. In the context of spermatogenesis, conditional knockout models with stage-specific Cre drivers allow precise temporal control of gene inactivation to determine when each EIF4G protein is required. Cross-rescue experiments testing whether overexpression of one family member can compensate for loss of another provide important insights into functional redundancy and specialization .

What emerging technologies might enhance our understanding of EIF4G3's role in translational regulation during spermatogenesis?

Emerging technologies offer promising avenues to deepen our understanding of EIF4G3's role in translational regulation during spermatogenesis. Single-cell multiomics approaches that simultaneously profile transcriptomes, proteomes, and translatomes from individual spermatogenic cells could reveal cell type-specific functions of EIF4G3 throughout spermatogenesis. Advanced imaging technologies like lattice light-sheet microscopy with adaptive optics would enable real-time tracking of EIF4G3 dynamics during meiotic progression in live cells or tissue explants. Cryo-electron tomography could visualize the molecular architecture of translation complexes containing EIF4G3 in different subcellular compartments, potentially revealing distinct complex compositions in nuclear versus cytoplasmic locations. CRISPR-based epigenome editing could investigate how chromatin modifications within the XY body influence EIF4G3 recruitment and function. Spatially resolved transcriptomics and proteomics methods would map the relationship between EIF4G3 localization and local translation activities within different nuclear and cytoplasmic domains. Synthetic biology approaches, such as optogenetic control of EIF4G3 localization or activity, could precisely manipulate its function in specific cellular compartments to determine causal relationships. Patient-derived induced pluripotent stem cells differentiated toward the germ cell lineage offer opportunities to study human-specific aspects of EIF4G3 function in spermatogenesis, particularly in the context of infertility-associated mutations .

How can researchers effectively study the relationship between EIF4G3 and miRNA-mediated translational regulation?

To effectively study the relationship between EIF4G3 and miRNA-mediated translational regulation, researchers should implement a multifaceted experimental approach. Begin by identifying potential regulatory miRNAs using bioinformatic prediction algorithms to scan the EIF4G3 transcript for miRNA binding sites. The connection to tumor suppressor miRNA miR-520c-3p mentioned in search results provides an initial candidate for investigation . Validate direct interactions through luciferase reporter assays with wild-type and mutated miRNA binding sites from the EIF4G3 3' UTR. For functional studies, overexpress or inhibit candidate miRNAs and assess effects on EIF4G3 expression levels and translation efficiency of target mRNAs. RNA immunoprecipitation (RIP) assays using antibodies against EIF4G3 and components of the miRNA machinery (Ago2) can identify mRNAs co-regulated by both pathways. CLIP-seq (crosslinking immunoprecipitation followed by sequencing) would provide transcriptome-wide maps of EIF4G3 binding sites in relation to miRNA target sites. To understand tissue-specific regulation, compare miRNA and EIF4G3 expression patterns across different tissues and developmental stages, with particular attention to spermatogenesis. For mechanistic insights, examine how miRNA-mediated regulation of EIF4G3 affects its interaction with other translation factors and its subcellular localization. In disease contexts like the mentioned diffuse large B cell lymphoma (DLBCL) where EIF4G3 is upregulated, investigate whether dysregulation of specific miRNAs contributes to this overexpression .