EIF4G2 Antibody

What is EIF4G2 and why is it important for research?

EIF4G2, also known by several synonyms including p97, DAP5, NAT1, and EIF4G2/p97, is a eukaryotic translation initiation factor that plays crucial roles in both cap-independent and cap-dependent translation mechanisms . Unlike other eIF4G family members (eIF4G1 and eIF4G3), EIF4G2 lacks binding sites for the 5' cap binding protein eIF4E and for PABP, giving it unique functional characteristics in translation regulation . It has gained significant research interest due to its involvement in internal ribosome entry sites (IRESs)-mediated translation, as well as its roles in apoptosis, cell survival, cell differentiation, and embryonic development . These diverse cellular functions make EIF4G2 an important research target for understanding fundamental cellular processes and potentially for therapeutic development.

What types of EIF4G2 antibodies are available for research applications?

Research-grade EIF4G2 antibodies are available in multiple formats with different characteristics:

• Polyclonal antibodies: Such as the EIF4G2 Rabbit Polyclonal Antibody (CAB2897), produced against recombinant fusion protein containing amino acids 320-490 of human EIF4G2 , and Cell Signaling Technology's antibody #2182 produced against synthetic peptide corresponding to residues surrounding Glu805 .

• Monoclonal antibodies: Including fluorescently conjugated versions like the CoraLite® Plus 488-conjugated mouse monoclonal antibody (CL488-67428) .

These antibodies differ in their specificities, applications, and detection methods, allowing researchers to select the most appropriate tool based on their specific experimental requirements.

What is the difference between EIF4G2 and other eIF4G family members in terms of antibody selection?

For antibody selection, this means:

• Researchers must carefully evaluate the epitope region recognized by the antibody to ensure specificity for EIF4G2 and not other family members

• Cross-reactivity testing is essential, particularly in applications where multiple eIF4G family members may be present

• For functional studies, antibodies targeting different domains may yield different results based on the specific interactions being disrupted

Both eIF4G1 and eIF4G3 are primarily involved in cap-dependent translation, while EIF4G2 has a broader role in various translation mechanisms, including IRES-mediated translation during cellular stress .

Experimental Applications and Protocols

For optimal Western blot detection of EIF4G2, consider the following methodological approach:

• Sample preparation:

  • Use RIPA or NP-40 buffer with protease inhibitors

  • For complete extraction of EIF4G2, which has cytosolic, nuclear, and membrane-associated populations, consider sequential extraction methods

• Gel selection and transfer conditions:

  • Use 8-10% SDS-PAGE gels to effectively resolve the 97 kDa EIF4G2 protein

  • Transfer to PVDF membranes at lower amperage (30-40mA) for longer duration (overnight) to ensure complete transfer of larger proteins

• Antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1-2 hours

  • Incubate with primary antibody at recommended dilutions (1:500-1:1000)

  • Validated positive controls include HeLa, 293T, Jurkat, and LO2 cell lysates

• Detection considerations:

  • Expected molecular weight is approximately 97 kDa

  • Verify specificity using knockdown or knockout controls

  • For phosphorylation studies, consider phosphatase treatment controls

This methodology enhances detection sensitivity and specificity while minimizing background interference.

How can I optimize immunofluorescence experiments for EIF4G2 subcellular localization studies?

For accurate subcellular localization studies of EIF4G2 using immunofluorescence:

• Fixation and permeabilization optimization:

  • Test both paraformaldehyde (4%, 10-15 minutes) and methanol fixation (100%, -20°C, 10 minutes)

  • For permeabilization, compare 0.1-0.5% Triton X-100 and 0.1-0.5% saponin to determine which better preserves EIF4G2 epitopes while allowing antibody access

• Antibody selection and dilution:

  • For direct visualization, the CoraLite® Plus 488-conjugated monoclonal antibody (CL488-67428) provides excellent signal with reduced background

  • For unconjugated antibodies, use dilutions between 1:50-1:200 for optimal signal-to-noise ratio

• Co-localization studies:

  • Combine with markers for specific subcellular compartments:

    • Adherens junctions: E-cadherin or β-catenin

    • Translation initiation complexes: eIF4A, eIF3

    • Stress granules: G3BP1, TIA-1

• Validation strategies:

  • Include EIF4G2 knockdown controls

  • Compare localization in stressed vs. unstressed conditions to observe translocation patterns

  • Use super-resolution microscopy for detailed co-localization analysis

This methodological approach enables accurate assessment of EIF4G2's dynamic subcellular distribution and potential functional interactions.

How do I address inconsistent Western blot results with EIF4G2 antibodies?

Inconsistent Western blot results when detecting EIF4G2 may stem from several experimental factors:

• Sample preparation issues:

  • EIF4G2 is susceptible to proteolytic degradation; ensure complete protease inhibition

  • Cell type-specific expression levels vary significantly; HeLa, 293T, Jurkat, and LO2 cells are validated positive controls

  • Stress conditions can alter EIF4G2 levels and post-translational modifications

• Antibody-specific considerations:

  • Different antibodies target distinct epitopes; the polyclonal antibody CAB2897 targets amino acids 320-490 , while Cell Signaling's antibody targets residues surrounding Glu805

  • Validate antibody lot-to-lot consistency with positive control lysates

  • For reproducibility, maintain consistent antibody incubation conditions (temperature, duration, concentration)

• Technical troubleshooting approaches:

  • If band intensity is weak, increase protein loading (50-80μg total protein)

  • For high background, increase blocking time and washing steps

  • Multiple bands may represent isoforms, post-translational modifications, or degradation products

  • Consider testing multiple antibodies targeting different epitopes to confirm identity

When inconsistencies persist, side-by-side comparison of different antibodies on the same samples can help identify the most reliable reagent for your specific experimental system.

What controls are essential for validating EIF4G2 antibody specificity?

Rigorous validation of EIF4G2 antibody specificity requires multiple complementary controls:

• Genetic manipulation controls:

  • siRNA or shRNA knockdown of EIF4G2 (partial reduction)

  • CRISPR/Cas9 knockout (complete elimination)

  • Overexpression of tagged EIF4G2 (band shift or increased signal)

• Peptide competition assays:

  • Pre-incubation of antibody with immunizing peptide should abolish specific signal

  • Non-competing peptides from different regions should not affect signal

• Cross-species reactivity assessment:

  • Test antibody performance across human, mouse, and rat samples when cross-reactivity is claimed

  • Document species-specific molecular weight variations

• Multiple antibody verification:

  • Compare results using antibodies targeting different epitopes

  • Correlation between results increases confidence in specificity

• Application-specific controls:

  • For immunoprecipitation: include IgG control

  • For immunofluorescence: include secondary-only controls and competition controls

Documentation of these validation experiments is increasingly required by journals and strengthens the reliability of research findings.

How can EIF4G2 antibodies be applied to study stress-induced translational reprogramming?

EIF4G2 plays critical roles in stress-induced translational reprogramming, particularly in mediating IRES-driven and other cap-independent translation mechanisms . Advanced research applications include:

• Stress granule association analysis:

  • Use co-immunoprecipitation with EIF4G2 antibodies to isolate stress-specific mRNP complexes

  • Combined with RNA-seq to identify stress-specific EIF4G2-associated transcripts

  • Compare EIF4G2-bound mRNAs under different stress conditions (oxidative, ER, nutrient deprivation)

• Translation state analysis:

  • Polysome profiling with EIF4G2 immunoblotting to track redistribution during stress

  • Ribosome profiling with EIF4G2 knockdown/overexpression to identify EIF4G2-dependent translation during stress

  • Cross-linking immunoprecipitation (CLIP) to map direct EIF4G2-mRNA interactions

• Mechanistic studies of cap-independent translation:

  • Use EIF4G2 antibodies to study recruitment to specific IRESs and CITE elements

  • Analyze stress-induced post-translational modifications of EIF4G2 that regulate its activity

  • Compare EIF4G2 and eIF4G1 recruitment to mRNAs during stress transitions

These approaches enable researchers to decipher how EIF4G2 contributes to selective mRNA translation during cellular stress, providing insights into stress adaptation mechanisms.

What approaches can resolve contradictory findings about EIF4G2's role in cap-dependent translation?

Recent research has challenged the traditional view of EIF4G2 as exclusively involved in cap-independent translation, with newer evidence suggesting roles in cap-dependent translation as well . To address these contradictions:

• Domain-specific functional analysis:

  • Use domain-specific antibodies to study interactions between EIF4G2 and other translation factors

  • Compare binding partners of EIF4G2 and eIF4G1 through differential immunoprecipitation

  • Analyze how specific domains contribute to different modes of translation initiation

• Context-dependent activity assessment:

  • Examine cell type-specific roles using tissue-specific antibody panels

  • Compare EIF4G2 function across developmental stages

  • Analyze how cellular conditions influence EIF4G2's participation in different translation modes

• Advanced integrative approaches:

  • Combine CRISPR-mediated tagging of endogenous EIF4G2 with proximity labeling

  • Use time-resolved immunoprecipitation during translation initiation to capture dynamic interactions

  • Implement single-molecule imaging with labeled antibodies to visualize EIF4G2 recruitment to different translation complexes

These methodologies can help reconcile seemingly contradictory findings by providing a more nuanced understanding of EIF4G2's context-dependent functions in translation regulation.

How can phospho-specific EIF4G2 antibodies inform understanding of translational regulation?

While standard EIF4G2 antibodies detect total protein levels, phospho-specific antibodies can provide critical insights into activity-dependent regulation. Researchers should consider:

• Phosphorylation site-specific analyses:

  • Several phosphorylation sites on EIF4G2 have been identified through proteomics

  • Phospho-specific antibodies allow tracking of specific modifications under different conditions

  • These modifications may dictate whether EIF4G2 participates in cap-dependent or -independent translation

• Kinase pathway studies:

  • Use phospho-specific antibodies to analyze how different signaling pathways modulate EIF4G2 activity

  • Compare phosphorylation patterns induced by stress, growth factors, and cell cycle progression

  • Determine how phosphorylation affects EIF4G2 protein interactions and subcellular localization

• Methodological considerations:

  • When using phospho-specific antibodies, always include phosphatase-treated controls

  • Consider dual staining with total and phospho-specific antibodies to calculate activation ratios

  • Use phospho-mimetic and phospho-null mutants as specificity controls

This advanced application of EIF4G2 antibodies can reveal the molecular switches that control its diverse functions in translation regulation.

How are EIF4G2 antibodies being utilized to study its role in embryonic development and cell differentiation?

EIF4G2 has been implicated in embryonic development and cell differentiation processes . Cutting-edge research applications include:

• Developmental expression profiling:

  • Immunohistochemical analysis of EIF4G2 expression across developmental stages

  • Co-staining with lineage markers to identify cell type-specific expression patterns

  • Comparison with other eIF4G family members to identify developmental switches

• Stem cell differentiation studies:

  • Track EIF4G2 levels and localization during directed differentiation protocols

  • Analyze correlation between EIF4G2 expression and translation of differentiation-specific mRNAs

  • Compare wild-type and EIF4G2-depleted cells during differentiation to identify stage-specific requirements

• Methodological approaches:

  • For developmental studies, optimize fixation conditions to preserve tissue architecture

  • Use multi-color immunofluorescence to correlate EIF4G2 with developmental markers

  • Consider tissue clearing techniques for 3D visualization of EIF4G2 expression patterns

These applications provide insights into how translational control through EIF4G2 contributes to developmental timing and cell fate decisions.

What technical considerations are important when using EIF4G2 antibodies for cancer research applications?

EIF4G2's involvement in cell survival and apoptosis regulation makes it particularly relevant for cancer research . Specialized considerations include:

• Tumor heterogeneity analysis:

  • Use immunohistochemistry with EIF4G2 antibodies on tissue microarrays to assess expression across tumor types

  • Correlate expression with clinical outcomes and therapeutic responses

  • Compare primary tumors with metastases to track expression changes during progression

• Therapeutic response monitoring:

  • Analyze EIF4G2 levels before and after treatment with translation-targeting drugs

  • Use phospho-specific antibodies to track signaling-dependent regulation during treatment

  • Combine with proliferation and apoptosis markers to correlate EIF4G2 function with cellular outcomes

• Application-specific optimization:

  • For FFPE samples, optimize antigen retrieval methods (citrate vs. EDTA, pH variations)

  • For circulating tumor cells, consider using flow cytometry with CL488-67428

  • For patient-derived xenografts, verify cross-reactivity between human EIF4G2 antibodies and mouse background

These approaches enable researchers to evaluate EIF4G2 as both a biomarker and potential therapeutic target in cancer.