EIF4G2 Antibody, HRP conjugated

What is EIF4G2 and what is its function in cellular processes?

EIF4G2 is a translation initiation factor that functions as a general repressor of translation by forming translationally inactive complexes. It appears to play a critical role in the switch from cap-dependent to IRES-mediated translation during mitosis, apoptosis, and viral infection. EIF4G2 interacts with protein kinases Mnk1 and Mnk2, as well as binding to EIF4A and EIF3 . Unlike the conventional eIF4G, which supports both cap-dependent and independent translation, EIF4G2 primarily forms complexes that inhibit translation initiation . Research has shown that EIF4G2 shares similarity with the C-terminal region of eIF4G containing binding sites for eIF4A and eIF3, but lacks the N-terminal binding site for eIF4E that is present in eIF4G .

What applications are EIF4G2 antibodies typically used for?

EIF4G2 antibodies are validated for multiple applications across molecular and cellular biology research:

These applications enable researchers to investigate EIF4G2 expression, localization, and interactions in various experimental contexts .

How should I optimize Western blot protocols specifically for HRP-conjugated EIF4G2 antibodies?

When optimizing Western blot protocols for HRP-conjugated EIF4G2 antibodies, start with a dilution range of 1:500 to 1:1000 for the primary antibody . For optimal results, load 20-40 μg of total protein lysate per lane and block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature. Since the antibody is directly conjugated to HRP, you can proceed directly to detection after primary antibody incubation and washing steps, skipping the secondary antibody incubation. Extended washing (4-5 washes of 5 minutes each) is crucial to reduce background signal with direct HRP conjugates. If signal intensity is low, consider extending the primary antibody incubation time to overnight at 4°C rather than increasing concentration, as this typically improves signal-to-noise ratio without increasing background .

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

A robust experimental design with EIF4G2 antibodies should include the following controls:

• Positive Control: Include lysates from cell lines known to express EIF4G2, such as HeLa, 293T, Jurkat, LO2, or mouse brain tissue .

• Negative Control: Consider using:

  • EIF4G2 knockdown or knockout cell lines

  • Blocking peptide control (pre-incubate antibody with immunizing peptide)

  • Isotype control antibody (rabbit IgG for rabbit polyclonal antibodies)

• Loading Control: Include detection of housekeeping proteins (β-actin, GAPDH, tubulin) to ensure equal loading across samples.

• Molecular Weight Marker: Confirm the detected band appears at the expected molecular weight of approximately 97-102 kDa .

For immunostaining experiments, include an unstained control and a secondary-only control (if using unconjugated primary antibodies) to assess background staining levels .

How can I optimize antigen retrieval for immunohistochemistry with EIF4G2 antibodies?

For optimal antigen retrieval when using EIF4G2 antibodies in immunohistochemistry of formalin-fixed, paraffin-embedded (FFPE) tissues, high-pressure antigen retrieval in citrate buffer (pH 6.0) has shown superior results . After dewaxing and hydration, sections should be subjected to this antigen retrieval method followed by blocking with 10% normal goat serum for 30 minutes at room temperature. For the primary antibody incubation, use a 1:50 to 1:200 dilution in 1% BSA and incubate overnight at 4°C to ensure optimal antigen binding while minimizing background staining. For visualization, use a biotinylated secondary antibody followed by an HRP-conjugated detection system, or if using an HRP-conjugated primary antibody, proceed directly to chromogenic visualization after thorough washing . This protocol has been successfully validated on multiple tissue types and provides consistent, reproducible results for EIF4G2 detection.

Why might I be observing multiple bands or non-specific binding with my EIF4G2 antibody?

Multiple bands or non-specific binding when using EIF4G2 antibodies can occur for several reasons:

• Alternative Splicing and Isoforms: EIF4G2 has multiple isoforms resulting from alternative splicing. Research has confirmed the existence of these isoforms with varying molecular weights, which may appear as multiple bands in Western blots .

• Post-translational Modifications: EIF4G2 undergoes various post-translational modifications, including phosphorylation and proteolytic cleavage. These modifications can alter the apparent molecular weight of the protein, resulting in additional bands. In particular, EIF4G2 is known to be cleaved by caspases during apoptosis and by viral proteases during infection .

• Cross-Reactivity: Some antibodies may cross-react with structurally similar proteins, especially other members of the eIF4G family. To minimize this issue, use antibodies raised against unique regions of EIF4G2 that don't share significant homology with other proteins.

• Degradation Products: Improper sample handling can lead to protein degradation, resulting in additional lower molecular weight bands. Ensure samples are properly prepared with protease inhibitors and kept cold throughout the preparation process .

To address these issues, try optimizing blocking conditions (5% milk vs. BSA), increasing the number and duration of washing steps, and further diluting the primary antibody to reduce non-specific binding.

What are the common pitfalls when using HRP-conjugated antibodies for Western blotting?

When using HRP-conjugated antibodies for Western blotting, researchers should be aware of these common pitfalls:

• Signal Saturation: HRP-conjugated primary antibodies can produce very strong signals, potentially leading to signal saturation. Start with shorter exposure times and adjust as needed to capture the linear range of signal intensity.

• Limited Shelf-Life: The HRP conjugation may reduce the shelf-life of the antibody compared to unconjugated versions. Store according to manufacturer recommendations, typically at -20°C, avoiding freeze/thaw cycles, and consider adding glycerol to a final concentration of 50% to prevent freezing damage .

• Temperature Sensitivity: HRP activity is temperature-dependent, so ensure consistent laboratory temperature during experiments to maintain reproducibility. Avoid exposing the conjugated antibody to temperatures above 4°C for extended periods.

• Endogenous Peroxidase Activity: Samples may contain endogenous peroxidase activity that can generate false-positive signals. Consider including a peroxidase quenching step (such as treatment with 0.3% H₂O₂ in methanol for 30 minutes) before blocking, especially in IHC applications.

• Sodium Azide Incompatibility: Never use sodium azide in buffers with HRP-conjugated antibodies, as it inhibits HRP activity. Double-check all buffer components before use .

• Overdevelopment: Extended development times can increase background signal. Monitor the development process carefully and stop the reaction at the appropriate time.

Following these precautions will help ensure reliable and consistent results with HRP-conjugated EIF4G2 antibodies.

How can I use EIF4G2 antibodies to investigate its role in cancer progression?

Recent research has revealed complex relationships between EIF4G2 expression and cancer progression that can be further investigated using EIF4G2 antibodies in several sophisticated experimental approaches:

These methods leverage EIF4G2 antibodies to provide mechanistic insights into how alterations in translation initiation factors contribute to cancer development and progression.

How can I investigate EIF4G2 protein interactions using co-immunoprecipitation?

To investigate EIF4G2 protein interactions using co-immunoprecipitation (co-IP), follow this optimized protocol:

• Cell Lysis: Lyse cells in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease/phosphatase inhibitors. For studying RNA-dependent interactions, consider parallel samples with and without RNase treatment.

• Pre-Clearing: Pre-clear lysates with protein A/G beads for 1 hour at 4°C to reduce non-specific binding.

• Immunoprecipitation: Add 0.5-4 μg of EIF4G2 antibody to 200-400 μg of pre-cleared lysate and incubate overnight at 4°C with gentle rotation . For the control IP, use the same amount of isotype-matched IgG.

• Bead Addition: Add protein A/G beads and incubate for 2-4 hours at 4°C.

• Washing: Perform 4-5 stringent washes with lysis buffer containing reduced detergent concentration (0.1-0.5% Triton X-100).

• Elution and Analysis: Elute bound proteins by boiling in SDS sample buffer and analyze by Western blotting for potential interacting partners.

This approach has been successfully used to identify EIF4G2 interactions with translation factors (like EIF4A and EIF3) and protein kinases (Mnk1 and Mnk2) . More advanced applications include combining co-IP with mass spectrometry to identify novel interaction partners, as demonstrated in studies of cancer-associated EIF4G2 mutations .

What are the implications of EIF4G2 mutations in disease contexts and how can they be studied?

EIF4G2 mutations have significant implications in disease contexts, particularly in cancer. Recent research has identified several loss-of-function cancer-associated mutations in EIF4G2, including R165C, R178Q, R295C, R714H, and N785K . These mutations affect the protein's ability to interact with its binding partners, potentially altering translation regulation pathways that contribute to disease progression.

To study these mutations effectively:

• Structural and Functional Analysis: Compare wild-type and mutant EIF4G2 using co-immunoprecipitation followed by mass spectrometry to identify altered protein interactions . This approach has revealed that certain mutations disrupt key protein-protein interactions essential for EIF4G2 function.

• Translation Efficiency Assessment: Measure the impact of EIF4G2 mutations on translation efficiency using polysome profiling or ribosome profiling, followed by Western blot analysis with EIF4G2 antibodies to correlate protein levels with translation status.

• CRISPR/Cas9 Gene Editing: Generate isogenic cell lines harboring specific EIF4G2 mutations using CRISPR/Cas9 technology, then use EIF4G2 antibodies to confirm expression levels and study downstream effects on cellular phenotypes.

• Patient-Derived Models: Analyze EIF4G2 expression and mutation status in patient-derived xenografts or organoids using immunohistochemistry and genomic sequencing to correlate with treatment responses and clinical outcomes .

These approaches provide complementary insights into how EIF4G2 mutations contribute to disease pathogenesis and may identify potential therapeutic vulnerabilities in cancers with altered EIF4G2 function.

How should I select between polyclonal and monoclonal EIF4G2 antibodies for my research?

Selecting between polyclonal and monoclonal EIF4G2 antibodies requires careful consideration of your specific research objectives:

Polyclonal Antibodies (e.g., rabbit polyclonal EIF4G2 antibodies):

• Advantages: Recognize multiple epitopes on EIF4G2, providing stronger signals even if some epitopes are masked or modified. This is particularly useful for detecting EIF4G2 in fixed tissues where protein folding may be altered .

• Best Applications: Immunohistochemistry of fixed tissues, detection of denatured proteins in Western blots, and initial screening studies.

• Considerations: Batch-to-batch variation may occur, potentially affecting experimental reproducibility over long-term studies.

Monoclonal Antibodies:

• Advantages: Recognize a single epitope, providing high specificity and reproducibility. Ideal for distinguishing between closely related proteins or specific isoforms of EIF4G2.

• Best Applications: Precise localization studies, differentiation between EIF4G2 isoforms, and long-term studies requiring consistent reagents.

• Considerations: May lose reactivity if the specific epitope is masked, modified, or destroyed during sample processing.

For most comprehensive studies, consider using both antibody types in complementary approaches. If detecting EIF4G2 across different species is important, verify cross-reactivity (human, mouse, and rat reactivity is confirmed for several commercially available antibodies) .

What are the optimal sample preparation methods for detecting EIF4G2 in different experimental contexts?

Optimal sample preparation for EIF4G2 detection varies by experimental context:

For Western Blotting:

• Lyse cells in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) with protease inhibitors.

• Sonicate briefly to shear DNA and reduce viscosity.

• Centrifuge at 14,000 × g for 15 minutes at 4°C to remove debris.

• Determine protein concentration using Bradford or BCA assay.

• Denature samples in Laemmli buffer at 95°C for 5 minutes.

• Load 20-40 μg protein per lane for optimal EIF4G2 detection .

For Immunohistochemistry (IHC-P):

• Fix tissues in 10% neutral buffered formalin for 24-48 hours.

• Process and embed in paraffin following standard protocols.

• Section tissues at 4-5 μm thickness.

• For antigen retrieval, use high-pressure method with citrate buffer (pH 6.0) .

• Block with 10% normal goat serum for 30 minutes at room temperature.

• Apply primary antibody diluted in 1% BSA and incubate overnight at 4°C .

For Immunofluorescence (IF/ICC):

• Fix cells in 4% formaldehyde for 15 minutes at room temperature.

• Permeabilize using 0.2% Triton X-100 for 10 minutes.

• Block in 10% normal goat serum for 1 hour.

• Apply EIF4G2 antibody at 1:50 to 1:200 dilution and incubate overnight at 4°C.

• For visualization, use fluorophore-conjugated secondary antibody or direct fluorophore-conjugated EIF4G2 antibody .

These optimized protocols ensure maximal detection sensitivity while maintaining specificity across different experimental applications.

How can I quantitatively analyze EIF4G2 expression levels in relation to disease progression?

Quantitative analysis of EIF4G2 expression in relation to disease progression requires rigorous methodological approaches:

These approaches have been successfully applied in studies of endometrial cancer, where EIF4G2 expression was analyzed in a cohort of 280 patients across different cancer types, grades, and stages, revealing significant correlations with patient outcomes over a 12-year follow-up period .