EIF4G2 Antibody

What is EIF4G2 and why is it important in research?

EIF4G2, also known as DAP5, p97, or NAT1, is a eukaryotic translation initiation factor that plays crucial roles in translation initiation and mRNA binding . Unlike canonical translation initiation factors, EIF4G2 is involved in non-canonical translation pathways, particularly during cellular stress responses where it facilitates the translation of selective mRNA cohorts . Its importance in research stems from its involvement in fundamental cellular processes like protein synthesis regulation and its implications in various diseases, including cancer . The protein has been observed with a molecular weight of approximately 97 kDa and comprises 907 amino acids . Understanding EIF4G2 function is essential for developing targeted therapies for conditions characterized by dysregulated protein synthesis.

What are the main types of EIF4G2 antibodies available for research?

Researchers have access to several types of EIF4G2 antibodies, each with specific applications and characteristics:

• Polyclonal antibodies (e.g., Rabbit Polyclonal Antibody CAB2897): These recognize multiple epitopes on the EIF4G2 protein, providing high sensitivity but potentially lower specificity .

• Monoclonal antibodies (e.g., 67428-1-Ig): These target a single epitope, offering high specificity and consistency between batches .

• Conjugated antibodies (e.g., CL488-67428): These come with fluorescent tags like CoraLite® Plus 488, allowing direct visualization in applications such as immunofluorescence microscopy and flow cytometry without requiring secondary antibodies .

The choice between these depends on the experimental application, with polyclonals often preferred for detection of native proteins, monoclonals for specific epitope targeting, and conjugated antibodies for direct visualization techniques.

How do I determine which species my EIF4G2 antibody will react with?

To confirm reactivity for your specific application:

• Check the "Tested Reactivity" section in the antibody datasheet, which lists species with confirmed reactivity through experimental validation .

• Review the immunogen sequence used to generate the antibody and compare it with the target protein sequence across species of interest to predict potential cross-reactivity .

• When possible, examine validation data in the form of Western blots, IHC, or IF images showing positive results with samples from your species of interest .

• If working with an unstudied species, consider conducting a preliminary validation experiment using positive control samples before proceeding with your main experiments.

What are the optimal dilution factors for different applications of EIF4G2 antibodies?

Optimal dilution factors vary by antibody and application. Based on manufacturer recommendations:

These dilutions serve as starting points, and optimization for specific experimental conditions is strongly recommended. Factors affecting optimal dilution include sample type, protein expression level, detection method sensitivity, and background signal levels in your experimental system.

How should I perform antigen retrieval when using EIF4G2 antibodies for IHC?

Effective antigen retrieval is crucial for successful immunohistochemistry with EIF4G2 antibodies. Based on manufacturer recommendations for the monoclonal antibody 67428-1-Ig, TE buffer at pH 9.0 is suggested as the primary antigen retrieval method . Alternatively, citrate buffer at pH 6.0 may be used if TE buffer doesn't yield optimal results.

A methodological approach to antigen retrieval for EIF4G2 IHC includes:

• Deparaffinize and rehydrate tissue sections through xylene and graded alcohols.

• Prepare fresh TE buffer (10mM Tris, 1mM EDTA, pH 9.0) or citrate buffer (10mM Sodium Citrate, pH 6.0).

• Immerse slides in the buffer within a heat-resistant container.

• Heat using one of the following methods:

  • Microwave: Heat to boiling, then maintain at sub-boiling temperature for 10-20 minutes

  • Pressure cooker: Process for 3-5 minutes at full pressure

  • Water bath: Incubate at 95-98°C for 20-30 minutes

• Allow slides to cool in the retrieval solution for 20 minutes at room temperature.

• Rinse thoroughly with PBS before proceeding with blocking and antibody incubation steps.

For optimal results, comparative testing of both TE and citrate buffer methods is recommended to determine which provides the best signal-to-noise ratio with your specific tissue samples.

What controls should I include when using EIF4G2 antibodies in my experiments?

Proper controls are essential for interpreting results with EIF4G2 antibodies. A comprehensive control strategy includes:

• Positive controls:

  • Cell lines with confirmed EIF4G2 expression such as HeLa, HEK-293, HepG2, Jurkat, LO2, and 293T cells

  • Tissue samples known to express EIF4G2, such as human prostate or breast cancer tissues

• Negative controls:

  • Primary antibody omission: Replace primary antibody with antibody diluent

  • Isotype control: Use matched isotype antibody (e.g., Rabbit IgG for polyclonal or Mouse IgG2a for monoclonal) at the same concentration

  • Blocking peptide: Pre-incubate antibody with the immunizing peptide to confirm specificity

• Knockdown/knockout validation:

  • Samples from EIF4G2 knockdown or knockout models to demonstrate antibody specificity

  • This is particularly important when studying EIF4G2 in new experimental contexts

• Loading controls (for Western blot):

  • Housekeeping proteins like GAPDH, β-actin, or α-tubulin to normalize for protein loading

  • Total protein staining methods like Ponceau S for membrane verification

Including these controls allows for proper validation of antibody specificity, correct interpretation of results, and troubleshooting of potential technical issues.

How can EIF4G2 antibodies be used to investigate its role in cancer progression?

Methodological approaches using EIF4G2 antibodies for cancer research include:

• Prognostic biomarker evaluation:

  • Use immunohistochemistry with validated EIF4G2 antibodies on tissue microarrays to correlate expression levels with patient outcomes

  • Quantify expression using digital pathology and image analysis software for objective scoring

  • Correlate with clinicopathological parameters and survival data over extended periods (5-12 years)

• Mechanistic investigations:

  • Employ Western blotting to assess EIF4G2 expression levels in cancer cell lines before and after treatments

  • Use immunoprecipitation (IP) to identify protein-protein interactions that may explain EIF4G2's role in cancer progression

  • Combine with siRNA/shRNA knockdown experiments to establish causative relationships between EIF4G2 levels and cancer cell behaviors

• Therapeutic resistance studies:

  • Monitor EIF4G2 expression changes in response to conventional therapies using immunofluorescence or flow cytometry

  • Correlate EIF4G2 levels with therapy resistance markers in patient-derived samples

  • Use conjugated antibodies for multiparameter flow cytometry to identify resistant cell populations

These approaches have revealed that EIF4G2 depletion can result in increased resistance to conventional therapies like Taxol and radiation treatment, and enrichment of aggressive cell subsets with high expression of CD133 and CD44 .

How do I resolve contradictory findings regarding EIF4G2 expression in different cancer types?

Contradictory findings regarding EIF4G2 expression across cancer types represent a significant challenge in the field. Recent literature suggests that EIF4G2's role may be context-dependent, with both decreased and increased expression observed in different malignancies .

To investigate and resolve these contradictions:

This methodical approach can help reconcile seemingly contradictory findings and establish a more nuanced understanding of EIF4G2's role in different cancer contexts.

What are the key considerations when using EIF4G2 antibodies to study translation regulation during cellular stress?

Studying translation regulation during cellular stress with EIF4G2 antibodies requires specific methodological considerations:

• Stress induction protocols:

  • Apply standardized stress conditions (e.g., hypoxia, nutrient deprivation, ER stress) with defined parameters

  • Include time-course analysis to capture dynamic changes in EIF4G2 function

  • Monitor stress markers concurrently to confirm successful stress induction

• Subcellular localization studies:

  • Use immunofluorescence with high-resolution microscopy to track EIF4G2 localization changes during stress

  • Co-stain with markers for stress granules, P-bodies, or other translation-related compartments

  • EIF4G2 has been localized to adherens junctions, cytosol, and eukaryotic translation initiation complexes

• Interaction partner analysis:

  • Employ co-immunoprecipitation with EIF4G2 antibodies to identify stress-specific binding partners

  • Confirm interactions with orthogonal methods (proximity ligation assay, FRET)

  • Compare interaction profiles between normal and stress conditions

• Translation target identification:

  • Combine EIF4G2 immunoprecipitation with RNA sequencing to identify bound mRNAs

  • Compare mRNA binding profiles under normal and stress conditions

  • Confirm translation of identified targets through proteomics approaches

  • Recent research identified kinesin-1 motor proteins (KIF5B and KLC1, 2, 3) as direct translation targets of EIF4G2

• Antibody selection considerations:

  • Choose antibodies recognizing epitopes that aren't masked during stress-induced conformational changes

  • For polyclonal antibodies like CAB2897, the recognition of multiple epitopes may provide more robust detection under varied conditions

  • For studies requiring high specificity, monoclonal antibodies may be preferable

These methodological considerations are essential for obtaining reliable and interpretable results when investigating EIF4G2's roles in stress-induced translation regulation.

Why might I experience high background when using EIF4G2 antibodies in immunofluorescence?

High background in immunofluorescence experiments with EIF4G2 antibodies can result from multiple factors. Addressing this issue requires a systematic approach:

• Antibody-related factors:

  • Excessive antibody concentration: Titrate the antibody to determine optimal concentration, starting from the manufacturer's recommended dilution range (e.g., 1:50-1:500 for conjugated antibodies or 1:400-1:1600 for unconjugated monoclonal antibodies)

  • Non-specific binding: Include additional blocking steps with 5% normal serum from the same species as the secondary antibody

  • For conjugated antibodies like CL488-67428, photobleaching or auto-fluorescence may contribute to background

• Sample preparation issues:

  • Incomplete fixation: Optimize fixation time and fixative concentration

  • Over-fixation: Excessive cross-linking can increase autofluorescence and non-specific binding

  • Inadequate permeabilization: Adjust detergent concentration and incubation time to ensure antibody access to the target

• Technical optimizations:

  • Include 0.1-0.3% Triton X-100 in antibody dilution buffers to reduce non-specific membrane binding

  • Add 0.1-0.5% BSA to all wash buffers to minimize non-specific interactions

  • For tissues, treat with autofluorescence reducers like sodium borohydride or specialized commercial reagents

  • When using fluorescent-conjugated antibodies like CL488-67428, minimize exposure to light during all steps

• Controls for troubleshooting:

  • Secondary antibody-only control to identify non-specific secondary antibody binding

  • Isotype control to identify Fc receptor-mediated binding

  • Pre-adsorption of the antibody with purified antigen to confirm specificity

Following these optimization steps systematically can significantly improve signal-to-noise ratio in EIF4G2 immunofluorescence experiments.

What are the most effective strategies for validating EIF4G2 antibody specificity?

Comprehensive validation of EIF4G2 antibody specificity is critical for ensuring reliable experimental results. Effective validation strategies include:

• Genetic approach validation:

  • siRNA/shRNA knockdown: Compare antibody signal in control versus EIF4G2-depleted samples

  • CRISPR/Cas9 knockout: Generate complete EIF4G2 knockout cells/tissues as the most stringent negative control

  • Overexpression: Test antibody in systems with controlled EIF4G2 overexpression to confirm signal increase

• Biochemical validation:

  • Western blot analysis: Confirm single band of expected molecular weight (approximately 97 kDa for EIF4G2)

  • Immunoprecipitation followed by mass spectrometry: Verify that the precipitated protein is indeed EIF4G2

  • Pre-adsorption test: Pre-incubate antibody with purified antigen or immunizing peptide before application to sample

• Comparative antibody validation:

  • Test multiple antibodies targeting different epitopes of EIF4G2

  • Compare polyclonal (e.g., CAB2897) versus monoclonal (e.g., 67428-1-Ig) antibody results

  • Validate across multiple applications (WB, IHC, IF) to confirm consistent target recognition

• Species and sample-type validation:

  • Test antibody in known positive samples from different species (human, mouse, rat)

  • Validate across different cell lines known to express EIF4G2 (e.g., HeLa, 293T, Jurkat, HepG2)

  • For tissue work, validate in multiple tissue types with different fixation and processing methods

Implementing these validation strategies before commencing extensive studies will ensure confidence in antibody specificity and experimental results.

How can I optimize EIF4G2 antibody-based detection in samples with low EIF4G2 expression?

Detecting low levels of EIF4G2 expression requires specialized techniques to enhance sensitivity without increasing background. Optimized strategies include:

• Signal amplification methods:

  • Tyramide Signal Amplification (TSA): Can increase detection sensitivity by 10-100 fold for IHC/IF applications

  • Biotin-streptavidin systems: Use biotinylated secondary antibodies followed by streptavidin-conjugated reporters

  • Polymer-based detection systems: Employ secondary antibodies conjugated to enzyme-labeled polymers for enhanced signal

• Technical optimizations for Western blot:

  • Increase protein loading (50-100μg total protein)

  • Use high-sensitivity ECL substrates or fluorescent secondary antibodies

  • Longer exposure times balanced against background increase

  • PVDF membranes (rather than nitrocellulose) for higher protein binding capacity

  • Consider using the more sensitive monoclonal antibody (67428-1-Ig) at its lower dilution range (1:5000)

• Immunoprecipitation enrichment:

  • Perform immunoprecipitation with the EIF4G2 antibody to concentrate the protein before Western blot detection

  • Use optimized IP conditions: 0.5μg-4μg antibody for 200μg-400μg extracts

  • Consider crosslinking to stabilize transient interactions

• Microscopy enhancements for IF/IHC:

  • Use confocal microscopy with increased laser power and detector gain

  • Extended primary antibody incubation (overnight at 4°C)

  • Consider the conjugated antibody (CL488-67428) at its lower dilution range (1:50) for direct visualization

  • Use antigen retrieval methods optimized for low-abundance proteins (high-pH EDTA buffer)

• Blocking and background reduction:

  • Extended blocking (2+ hours) with 5-10% serum or commercial blocking reagents

  • Addition of 0.1-0.3% Triton X-100 in antibody dilution buffers

  • Include 0.1-0.5% BSA in all wash buffers

These enhanced detection methods have proven particularly valuable in studies of endometrial cancer tissues where low EIF4G2 expression was associated with poor prognosis .

How can EIF4G2 antibodies be used to investigate translational reprogramming in cancer treatment resistance?

EIF4G2 antibodies provide powerful tools for investigating translational reprogramming associated with treatment resistance. Recent research has revealed that EIF4G2 depletion results in increased resistance to conventional therapies like Taxol and radiation treatment .

Methodological approaches include:

• Therapy response monitoring:

  • Use Western blot with EIF4G2 antibodies to measure expression changes before and after treatment

  • Perform immunohistochemistry on patient samples at baseline and post-treatment to correlate EIF4G2 levels with clinical outcomes

  • Combine with markers of therapy resistance to establish correlation or causation

• Cell population analysis:

  • Employ flow cytometry with conjugated EIF4G2 antibodies like CL488-67428 to identify and sort cell subpopulations based on EIF4G2 expression levels

  • Co-stain with markers of aggressive cell subsets (e.g., CD133, CD44) to identify resistant populations

  • Use this approach to isolate cells for further molecular characterization

• Mechanistic studies:

  • Conduct immunoprecipitation with EIF4G2 antibodies followed by mass spectrometry to identify differentially bound proteins or mRNAs in resistant versus sensitive cells

  • Perform comparative transcriptomic and proteomic analyses of EIF4G2-depleted cells to identify alterations in molecular pathways

  • Recent research identified decreased abundance of kinesin-1 motor proteins (KIF5B and KLC1, 2, 3) in EIF4G2-depleted cells, which correlated with poor survival in certain EC patients

• Therapeutic targeting assessment:

  • Use EIF4G2 antibodies to monitor expression after experimental therapeutics targeting translation

  • Combine with viability assays to correlate EIF4G2 levels with treatment efficacy

  • Develop co-culture models with mixed populations of cells expressing different levels of EIF4G2 to study competitive advantages under therapeutic pressure

These approaches can reveal the complex relationship between EIF4G2 expression and treatment resistance, potentially leading to improved patient stratification and therapeutic interventions.

What is the significance of using multiplexed imaging with EIF4G2 antibodies in cancer research?

Multiplexed imaging with EIF4G2 antibodies represents an advanced approach for understanding the complex relationships between EIF4G2 expression and other molecular markers in the tumor microenvironment. This technique has shown particular value in recent endometrial cancer research .

Key methodological considerations and applications include:

• Co-expression analysis with prognostic markers:

  • Simultaneously visualize EIF4G2 and its downstream targets (e.g., kinesin proteins) in the same tissue section

  • Correlate spatial expression patterns with tumor regions showing invasion or therapy resistance

  • Recent research employed multiplexed imaging to show correlation between decreased expression of kinesin proteins and poor survival in specific EC grades and stages

• Technical approaches for multiplexing:

  • Sequential immunofluorescence: Strip and re-probe the same section with different antibodies

  • Spectral unmixing: Use fluorophores with distinct spectra (e.g., combining CL488-67428 with other compatible fluorescent antibodies)

  • Mass cytometry imaging: Label antibodies with metal isotopes for highly multiplexed analysis

  • Cyclic immunofluorescence: Iterative imaging cycles with fluorophore inactivation between rounds

• Spatial relationship analysis:

  • Assess co-localization of EIF4G2 with translation machinery components

  • Determine cellular and subcellular distribution patterns in relation to cell signaling markers

  • Map EIF4G2 expression in relation to tumor-infiltrating immune cells or stromal components

• Integration with patient outcome data:

  • Correlate spatial patterns of EIF4G2 and interacting partners with survival data

  • Develop predictive signatures based on multiplexed marker patterns

  • Identify patient subgroups with distinct molecular signatures for potential therapeutic stratification

The power of this approach was demonstrated in recent research where multiplexed imaging of EC patient tumors revealed correlations between decreased expression of kinesin proteins and poor survival in specific grades and stages of cancer .

How might advances in antibody technology impact future EIF4G2 research?

Emerging antibody technologies promise to enhance EIF4G2 research in several key areas:

• Single-cell and subcellular resolution techniques:

  • Single-cell Western blot: Will allow assessment of EIF4G2 expression heterogeneity within tumor populations

  • Super-resolution microscopy with nanobodies: Smaller antibody fragments will enable visualization of EIF4G2 within crowded molecular complexes at nanometer resolution

  • Proximity labeling: Antibody-enzyme fusions that label neighboring proteins will help map the dynamic EIF4G2 interactome

• Intrabody applications:

  • Development of intracellularly expressed antibodies against EIF4G2 will allow real-time monitoring of endogenous EIF4G2 in living cells

  • This could reveal dynamic changes in EIF4G2 localization during stress responses and translation regulation

  • Combined with fluorescent reporters, these could track EIF4G2 activity in tumor xenografts in vivo

• Therapeutic antibody development:

  • The discovery that low EIF4G2 expression correlates with poor outcomes in certain cancers suggests potential for therapeutic antibodies that modulate EIF4G2 function

  • Antibody-drug conjugates targeting cancer cells with specific EIF4G2 expression patterns

  • Bispecific antibodies linking EIF4G2-expressing cells to immune effectors

• Multiomics integration platforms:

  • Antibody-based cellular indexing for simultaneous protein and mRNA profiling

  • Integration of EIF4G2 antibody-based proteomics with transcriptomics and genomics

  • This could help resolve contradictory findings regarding EIF4G2 expression across cancer types

• Enhanced validation methodologies:

  • Automated high-throughput antibody validation platforms

  • CRISPR-engineered cell lines for antibody validation

  • These approaches will increase confidence in research findings by ensuring antibody specificity

These technological advances will likely accelerate our understanding of EIF4G2's role in normal physiology and disease, potentially leading to new diagnostic and therapeutic approaches.