EIF4G3 Antibody, HRP conjugated

What is EIF4G3 and what cellular functions does it serve?

EIF4G3 (Eukaryotic translation initiation factor 4 gamma 3) functions as an important scaffold protein in the translation initiation complex. It is part of the eukaryotic translation initiation 4F (EIF4F) complex, a conserved mRNA cap-binding complex that mediates the first, rate-limiting step of translation initiation. This complex assembles on the 7-methylguanosine cap structure of mRNAs to facilitate the formation of a mRNA "closed loop" by interacting with poly(A)-binding protein (PABP) and the 5' cap . Beyond its canonical role in translation, EIF4G3 has been implicated in nuclear mRNA biogenesis and surveillance . Most notably, mutation of the Eif4g3 gene in mice causes male infertility with arrest of meiosis at the end of meiotic prophase, suggesting a highly specific role in spermatogenesis .

The EIF4F complex, of which EIF4G3 is a component, accomplishes several key functions during translation initiation:

• Unwinding local secondary structure in the 5' untranslated region of mRNA substrates

• Recruiting additional translation factors and the 40S ribosomal subunit

• Eventually being replaced on the mRNA by the translation elongation complex

What are the key specifications of commercially available EIF4G3 antibodies?

Commercial EIF4G3 antibodies are available in various formats with specific characteristics as outlined in the table below:

Research applications of EIF4G3 antibodies include Western blot, immunoprecipitation, immunohistochemistry, and immunofluorescence across human cell lines including A431, HEK-293, A549, HeLa, and HepG2 cells .

What is the significance of HRP conjugation for EIF4G3 antibody applications?

While the search results don't specifically detail HRP-conjugated EIF4G3 antibodies, HRP (horseradish peroxidase) conjugation provides significant advantages in numerous applications:

• Enhanced sensitivity: HRP enzymatically amplifies detection signals, enabling visualization of low-abundance proteins like EIF4G3 in complex biological samples.

• Simplified workflow: Direct HRP conjugation eliminates the need for secondary antibody incubation, reducing protocol time and potential sources of background.

• Quantitative detection: HRP-conjugated antibodies provide consistent signal-to-noise ratios, making them suitable for quantitative analyses in Western blots and ELISAs.

• Versatile detection methods: HRP-conjugated antibodies are compatible with multiple substrates including chemiluminescent, colorimetric, and fluorescent platforms.

• Multiplexing capability: When using substrates with different spectral properties, HRP-conjugated antibodies can be combined with other detection systems for simultaneous analysis of multiple targets.

What are the recommended dilutions and conditions for using EIF4G3 antibodies?

Optimal dilutions for EIF4G3 antibodies vary by application type and specific antibody preparation. Based on published data, the following ranges are recommended:

For specialized tissues like testes, where EIF4G3 shows specific nuclear localization, a 1:100 dilution has been successfully used in immunofluorescence applications . For HRP-conjugated versions, dilutions typically trend toward the higher end of these ranges (more dilute) due to the signal amplification provided by the enzymatic activity.

How should I design Western blot protocols for optimal EIF4G3 detection?

When designing Western blot protocols for EIF4G3 detection, consider these technical recommendations:

• Sample preparation:

  • Use RIPA or NP-40 based lysis buffers with protease inhibitors

  • For nuclear EIF4G3 detection, ensure proper nuclear extraction protocols are followed

• Gel selection:

  • Use 6-8% polyacrylamide gels due to the large size of EIF4G3 (calculated 177 kDa)

  • Consider gradient gels (4-15%) to better resolve potential isoforms

• Key considerations:

  • Be prepared to observe bands at both 70 kDa and 250 kDa rather than the calculated 177 kDa

  • Include positive control lysates from A431, HEK-293, A549, HeLa, or HepG2 cells

  • Block with 5% bovine serum albumin to reduce background

• For HRP-conjugated antibody protocol:

  • After transfer, block membrane with 5% BSA in TBST for 1 hour at room temperature

  • Incubate with HRP-conjugated EIF4G3 antibody at appropriate dilution overnight at 4°C

  • Wash extensively (4-5 times) with TBST

  • Develop using ECL substrate and detect signal via imaging system

  • No secondary antibody step required

What are the optimal methods for visualizing EIF4G3 in cells and tissues?

For cellular and tissue localization of EIF4G3, immunofluorescence and immunohistochemistry have revealed important biological insights. The following methodologies have proven successful:

For cellular immunofluorescence:

• Fix cells with 2-4% paraformaldehyde for 10-30 minutes

• Permeabilize with 0.3% Triton X-100 in PBS for 10-15 minutes

• Block with 5% bovine serum albumin for 30 minutes

• Apply primary EIF4G3 antibody (1:100-1:800 dilution depending on the antibody)

• Detect with fluorophore-conjugated secondary antibodies (1:500 Alexa Fluor 488 or 594)

For whole-mount or spread preparations of spermatocytes:

• Place cells on poly-L-lysine coated slides for 30 minutes

• Fix with 4% paraformaldehyde for 30 minutes

• Permeabilize with 0.3% Triton X-100 for 15 minutes

• Block and stain as described above

When examining spermatocytes, be prepared to observe an unexpected nuclear localization of EIF4G3, particularly its enrichment in the XY body (the chromatin domain formed by inactive sex chromosomes) . This unique localization pattern has significant implications for understanding EIF4G3's role in meiosis and spermatogenesis.

Why do I observe different molecular weights for EIF4G3 in Western blots?

The discrepancy between EIF4G3's calculated molecular weight (177 kDa) and observed weights (70 kDa and/or 250 kDa) can be explained by several biological and technical factors:

• Post-translational modifications: EIF4G3 may undergo tissue-specific phosphorylation, ubiquitination, or proteolytic processing that alters its apparent molecular weight.

• Alternative splicing: Multiple isoforms of EIF4G3 may be expressed in different tissues or developmental stages. The mouse Eif4g3 gene contains 26 exons and undergoes alternative splicing to produce different transcripts .

• Protein complexes: The 250 kDa band may represent EIF4G3 in stable complexes with other proteins that remain partially associated even under denaturing conditions.

• Proteolytic cleavage: The 70 kDa band may represent specific cleavage products that retain the epitope recognized by the antibody.

To confirm band identity, consider these validation approaches:

• siRNA knockdown of EIF4G3

• Use of Eif4g3 knockout or conditional knockout samples as negative controls

• Comparison of band patterns across different EIF4G3 antibodies recognizing different epitopes

• Mass spectrometry analysis of immunoprecipitated protein bands

How can I optimize signal specificity when working with EIF4G3 antibodies?

To enhance signal specificity when using EIF4G3 antibodies:

• Validate antibody specificity:

  • Test antibody on lysates from cells with EIF4G3 knockdown

  • Consider using Eif4g3 conditional knockout mice samples as negative controls

  • Compare results across multiple antibodies targeting different EIF4G3 epitopes

• Optimize blocking and washing:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Increase washing stringency with higher detergent concentrations or extended wash times

  • Consider pre-adsorbing antibodies against non-specific proteins

• Dilution optimization:

  • Titrate antibody concentrations to identify optimal signal-to-noise ratios

  • For HRP-conjugated antibodies, start with higher dilutions (1:2000-1:5000) and adjust as needed

• For immunostaining applications:

  • Include peptide competition controls

  • Carefully optimize fixation and permeabilization protocols

  • Use antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0 for tissue sections

What insights have EIF4G3 antibodies revealed about nuclear translation regulation?

EIF4G3 antibody studies have revealed surprising insights about nuclear localization and potential functions:

One of the most unexpected findings is that EIF4G3 localizes to the nucleus of spermatocytes, where it is highly enriched in the XY body, the chromatin domain formed by the transcriptionally inactive sex chromosomes . This observation challenges the conventional view that translation initiation factors function exclusively in the cytoplasm.

Furthermore, many other (but not all) translation-related proteins also localize in the XY body . This unexpected nuclear localization suggests potential roles beyond canonical translation:

• Nuclear mRNA metabolism: EIF4G3 may participate in nuclear mRNA processing, surveillance, or export pathways .

• "Poising" of translation complexes: Translation components in the XY body may be prepared or "poised" for rapid deployment during the meiotic division phase .

• Regulatory functions: Nuclear EIF4G3 may regulate specific transcripts critical for meiosis progression, such as Hspa2, which encodes heat-shock chaperone protein HSPA2 .

These observations potentially connect the XY body to post-transcriptional regulation during spermatogenesis, suggesting novel mechanisms for controlling gene expression during this critical developmental process.

How can EIF4G3 antibodies contribute to male fertility research?

EIF4G3 antibodies have become valuable tools in understanding male fertility mechanisms:

• Phenotypic connections: Mutations in the Eif4g3 gene cause male infertility in mice, with arrest of meiosis at the end of meiotic prophase, but no other obvious phenotypes . This makes EIF4G3 a highly specific factor in male reproductive biology.

• Molecular mechanisms: EIF4G3 antibodies have helped identify downstream targets like Hspa2, which encodes the heat-shock chaperone protein HSPA2. The translation of Hspa2 is blocked by mutation of the Eif4g3 gene . HSPA2 is required for activation of Maturation Promoting Factor (MPF), promoting the transition to the division phase .

• Subcellular localization insights: The unexpected nuclear localization of EIF4G3 in spermatocytes, particularly in the XY body, suggests specialized regulatory mechanisms during meiosis .

• Translational regulation: EIF4G3 may control the translation of specific mRNAs critical for meiotic progression and spermatid development, representing a post-transcriptional regulatory layer in spermatogenesis .

Researchers studying male fertility should consider EIF4G3 antibodies for:

• Identifying stage-specific expression patterns during spermatogenesis

• Investigating interactions with other fertility-related proteins

• Examining potential biomarkers for male infertility diagnosis

• Evaluating translation regulation in models of impaired spermatogenesis

What techniques can be used to study EIF4G3's role in translation initiation complexes?

To investigate EIF4G3's role in translation initiation complexes, researchers can employ several advanced techniques:

• Co-immunoprecipitation (Co-IP):

  • Use EIF4G3 antibodies to pull down associated proteins

  • Identify interaction partners by Western blot or mass spectrometry

  • HRP-conjugated antibodies can simplify detection in Western blot validation

• Proximity ligation assay (PLA):

  • Visualize direct interactions between EIF4G3 and other translation factors

  • Quantify interactions in different cellular compartments, including the nucleus

  • Particularly useful for studying the unexpected nuclear localization in spermatocytes

• Puromycin incorporation assay:

  • Detect active translation sites using puromycin and anti-puromycin antibodies

  • Co-stain with EIF4G3 antibodies to correlate localization with translation activity

  • This approach has been successfully applied to germ cells

• FRAP (Fluorescence Recovery After Photobleaching):

  • Study dynamics of EIF4G3-containing complexes in living cells

  • Analyze recruitment kinetics of EIF4G3 to different cellular compartments

• Quantitative expression analysis:

  • Use RT-PCR methods to correlate EIF4G3 mRNA levels with protein levels

  • Compare expression across tissues and developmental stages

• Targeted proteomics:

  • Identify EIF4G3-dependent changes in the translation of specific proteins

  • Compare wildtype and Eif4g3 mutant samples to identify affected substrates

How does EIF4G3 localization in the XY body impact experimental design?

The unexpected finding that EIF4G3 localizes to the XY body in spermatocytes has important implications for experimental design:

• Subcellular fractionation considerations:

  • Standard cytoplasmic extraction protocols may miss significant nuclear pools of EIF4G3

  • Include nuclear fractions when analyzing EIF4G3 in testicular tissues

  • Compare nuclear and cytoplasmic distribution across cell types and developmental stages

• Immunofluorescence strategy:

  • Include co-staining with XY body markers (such as γH2AX or SUMO-1)

  • Optimize fixation and permeabilization for nuclear antigen preservation

  • Use confocal microscopy to accurately assess nuclear localization patterns

• Functional analysis approaches:

  • Design experiments to investigate both cytoplasmic and nuclear functions

  • Consider chromatin immunoprecipitation to identify potential DNA interactions

  • Evaluate effects of EIF4G3 depletion on both cytoplasmic translation and nuclear processes

• Temporal considerations:

  • EIF4G3 localization may change during meiotic progression

  • Stage-specific analyses are essential for comprehensive understanding

  • Use synchronized cell populations when possible

• Technical validation:

  • Include multiple EIF4G3 antibodies targeting different epitopes

  • Use Eif4g3 conditional knockout mice as negative controls

  • Compare localization patterns across species to determine evolutionary conservation

This unexpected nuclear localization suggests that experimental designs focusing solely on cytoplasmic translation functions of EIF4G3 may miss critical aspects of its biology, particularly in reproductive tissues.