EIF4H Antibody

What is EIF4H and what are its primary functions in translation?

EIF4H is a eukaryotic translation initiation factor that enhances the ATP-dependent helicase activity of eIF4A within the eIF4F complex. The protein functions during translation initiation by helping unwind inhibitory RNA secondary structures within the 5'UTR of mRNAs . This unwinding action increases the ability of the 40S ribosomal subunit to bind to mRNA, facilitating efficient translation initiation. EIF4H exists in two isoforms: a longer 27 kDa isoform (4HL) and a shorter 25 kDa isoform (4Hs), both of which have been shown to contribute to translation regulation . The protein's activity is particularly important for the translation of mRNAs with complex secondary structures or IRES elements.

What are the known isoforms of EIF4H and how do they differ in function?

EIF4H exists in two primary splice variants: a longer 27 kDa isoform (4HL) and a shorter 25 kDa isoform (4Hs) . Research has demonstrated that both isoforms can contribute to malignant transformation when overexpressed in NIH3T3 cells. Functional studies indicate that both isoforms can:

• Stimulate cellular transformation and invasion

• Enhance tumor growth

• Increase resistance to drug-induced apoptosis

• Promote translation of mRNAs containing IRES elements or structured 5'UTRs

Though both isoforms show oncogenic potential, certain experimental contexts may reveal subtle functional differences that researchers should consider when designing knockdown or overexpression studies.

What criteria should be considered when selecting an EIF4H antibody for specific research applications?

When selecting an EIF4H antibody, researchers should evaluate:

• Target epitope recognition: Different antibodies recognize distinct regions of EIF4H. Available options include:

  • Antibodies targeting N-terminal regions (AA 1-100)

  • Antibodies recognizing central domains (AA 31-130, AA 127-156, AA 149-198)

  • C-terminal targeted antibodies

• Isoform specificity: Determine whether the antibody recognizes both the 27 kDa and 25 kDa isoforms or is specific to one variant .

• Host species and clonality: Both polyclonal (typically rabbit-derived) and monoclonal (mouse-derived) options are available . Monoclonal antibodies offer higher specificity but potentially lower sensitivity compared to polyclonal options.

• Validated applications: Confirm antibody validation for your intended application:

  • Western blotting (validated dilutions: 0.04-0.4 μg/mL)

  • Immunohistochemistry (validated dilutions: 1:50-1:200)

  • Immunofluorescence (validated dilutions: 0.25-2 μg/mL)

• Cross-reactivity profile: Consider species reactivity patterns. Available antibodies demonstrate varying reactivity across species:

  • Human-specific antibodies

  • Antibodies with cross-reactivity to mouse, rat, dog, monkey, and other models

How can researchers validate the specificity of EIF4H antibodies in their experimental systems?

Comprehensive validation of EIF4H antibodies should include:

• Positive and negative control samples:

  • Cell lines with known EIF4H expression levels

  • EIF4H knockdown models using validated shRNA constructs (as demonstrated in A549 and HeLa cells)

  • EIF4H overexpression systems (stable transfectants as described for NIH3T3 cells)

• Western blot validation:

  • Confirm detection of expected molecular weight bands (25 kDa and 27 kDa)

  • Verify band disappearance in knockdown samples

  • Test for cross-reactivity with related proteins

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide before detection

  • Signal should diminish proportionally to peptide concentration

• Orthogonal validation approaches:

  • Compare staining patterns across different antibodies targeting distinct epitopes

  • Correlate protein detection with mRNA expression data

  • Utilize multiple detection methods (IF, IHC, WB) to confirm consistent patterns

What are the most appropriate fixation and staining protocols for EIF4H immunohistochemistry in various tissue types?

Optimal protocols for EIF4H immunohistochemistry have been established through extensive tissue microarray testing. Key considerations include:

• Fixation parameters:

  • Standard formalin fixation is compatible with most EIF4H antibodies

  • For immunofluorescence applications, 4% paraformaldehyde fixation for 15-20 minutes is recommended

• Antigen retrieval methods:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) works effectively for paraffin sections

  • For frozen sections, brief fixation followed by permeabilization with 0.1% Triton X-100 is sufficient

• Antibody dilution ranges:

  • For immunohistochemistry: 1:50-1:200 dilution range is recommended

  • For immunofluorescence: 0.25-2 μg/mL concentration has been validated

• Detection systems:

  • For lung carcinoma tissues, HRP-DAB based detection systems provide optimal results

  • For double-labeling with angiogenesis markers (CD31, α-SMA), fluorescent secondary antibodies with minimal cross-reactivity should be selected

• Controls:

  • Include sections from tissues with known EIF4H expression patterns

  • Incorporate antibody omission controls to assess background staining

How can EIF4H antibodies be utilized to investigate translational regulation in cancer cells?

EIF4H antibodies serve as powerful tools for investigating translational control mechanisms in cancer:

• Polysome profiling:

  • EIF4H antibodies can identify shifts in EIF4H association with translating polysomes versus monosomes

  • Changes in this association pattern under drug treatment can reveal mechanisms of translational adaptation

• RNA immunoprecipitation (RIP):

  • EIF4H antibodies can be used to isolate mRNAs specifically bound to EIF4H

  • This approach has identified preferential binding to mRNAs encoding cell-proliferation factors (c-Myc, cyclin D1), angiogenic factors (FGF-2), and anti-apoptotic factors (CIAP-1, BCL-xL)

• Investigation of cancer-specific translational targets:

  • EIF4H knockdown has been shown to reduce expression of multiple FGF-2 isoforms and VEGF

  • Antibodies can be employed in co-immunoprecipitation studies to identify protein-protein interactions within the translation initiation complex under various stress conditions

• Visualization of stress granule formation:

  • Immunofluorescence with EIF4H antibodies can track redistribution of translation factors during cellular stress

  • This approach helps identify mechanisms of translation reprogramming during chemotherapy

What methodological approaches can resolve data inconsistencies when studying EIF4H in different experimental models?

When facing inconsistent results across experimental systems, consider these methodological solutions:

• Isoform-specific analysis:

  • Both 25 kDa and 27 kDa isoforms may show different expression patterns across cell types

  • Western blotting with high-resolution gels can distinguish these closely sized variants

  • Confirm antibody detection capabilities for both isoforms

• Cell line heterogeneity considerations:

  • EIF4H exhibits distinct functional consequences in different cell backgrounds

  • Compare results between multiple cell lines (A549, HeLa, NIH3T3) to identify consistent patterns

  • Document passage number and culture conditions in all experiments

• Context-dependent function assessment:

  • EIF4H function is most evident under specific stressors:

    • Serum deprivation (low serum conditions enhance proliferation differences)

    • Drug treatment (cisplatin or etoposide reveal survival phenotypes)

    • In vivo tumorigenic models demonstrate more pronounced effects than in vitro systems

• Quantitative considerations:

  • Standardize antibody dilutions across experiments

  • Employ quantitative image analysis for immunohistochemistry/immunofluorescence

  • Use calibrated protein standards for western blot quantification

How can researchers effectively characterize the interaction between EIF4H and eIF4A using immunological approaches?

The functional interaction between EIF4H and eIF4A is critical for translation initiation and can be investigated through:

• Co-immunoprecipitation strategies:

  • Using EIF4H antibodies to pull down protein complexes followed by eIF4A detection

  • Crosslinking approaches can stabilize transient interactions

  • Detergent optimization is crucial as interaction may be sensitive to extraction conditions

• Proximity ligation assays (PLA):

  • This technique allows visualization of protein-protein interactions in situ

  • Combining EIF4H antibodies with eIF4A antibodies from different host species

  • Quantification of PLA signals can reveal changes in interaction frequency under various conditions

• Biochemical helicase activity assays:

  • EIF4H antibodies can be used to immunodeplete the protein from cell extracts

  • Subsequent measurement of eIF4A helicase activity with synthetic RNA substrates

  • Reconstitution experiments with purified proteins confirm direct enhancement effects

• FRET-based interaction studies:

  • Antibody-based FRET pairs can assess dynamic interactions in live cells

  • This approach reveals spatial and temporal regulation of the EIF4H-eIF4A complex

What is the predictive value of EIF4H expression for chemotherapy response in different cancer types?

EIF4H expression patterns show promising potential as predictive biomarkers for chemotherapy response:

• Lung carcinoma chemoresistance correlation:

  • Immunohistochemical analysis of small cell lung carcinoma tissue microarrays revealed significant associations between EIF4H expression and treatment response

  • Among patients showing no objective response to etoposide and cisplatin therapy, approximately 80% exhibited positive EIF4H staining

  • Conversely, 58.1% of patients with objective response showed negative EIF4H staining

• Mechanistic basis for predictive capacity:

  • EIF4H overexpression inhibits drug-induced apoptotic pathways

  • Enhanced translation of anti-apoptotic factors (CIAP-1, BCL-xL) provides molecular explanation

  • These effects were consistently observed across multiple experimental models

• Standardization requirements:

  • Antibody-based detection protocols must be standardized for reproducible clinical assessment

  • Scoring systems for staining intensity require validation against clinical outcomes

  • Cut-off values for "positive" versus "negative" expression need prospective validation

How can EIF4H antibodies be optimized for multiplex immunofluorescence to study tumor microenvironment interactions?

Multiplex immunofluorescence strategies using EIF4H antibodies can reveal crucial interactions within the tumor microenvironment:

• Antibody panel design considerations:

  • Select EIF4H antibodies from host species compatible with other markers of interest

  • Validate each antibody individually before attempting multiplexing

  • Consider tyramide signal amplification for low-abundance targets

• Key marker combinations:

  • EIF4H + CD31 (endothelial marker): Reveals relationships between EIF4H expression and angiogenesis

  • EIF4H + α-SMA (pericyte marker): Assesses vascular maturation in relation to translational regulation

  • EIF4H + proliferation markers (Ki-67): Connects translational activity to cellular proliferation

  • EIF4H + apoptosis markers: Evaluates therapy response mechanisms

• Sequential staining approaches:

  • Antibody stripping or quenching between rounds enables use of antibodies from the same species

  • Spectral unmixing algorithms can resolve overlapping fluorophore emissions

  • Tissue autofluorescence must be accounted for, particularly in lung tissue specimens

• Image analysis strategies:

  • Cell segmentation algorithms can quantify marker co-expression at single-cell resolution

  • Spatial analysis reveals patterns of EIF4H expression relative to vascular structures

  • Correlation with clinical outcomes requires standardized image acquisition parameters

What are the methodological considerations for studying EIF4H-mediated translational control in patient-derived xenograft models?

Patient-derived xenograft (PDX) models offer unique opportunities to study EIF4H function in preserved tumor heterogeneity contexts:

• Antibody validation in xenograft tissues:

  • Confirm species specificity to distinguish human tumor cells from mouse stromal components

  • Validate epitope conservation in PDX models through western blot confirmation

  • Optimize antigen retrieval for potentially different fixation conditions

• Experimental design for therapy response studies:

  • Implement immunohistochemical assessment of EIF4H before and after treatment

  • Correlate expression patterns with therapy response metrics

  • Consider serial sampling approaches to track dynamic changes

• Translational readouts in PDX systems:

  • Use EIF4H antibodies in combination with phospho-specific antibodies for translation factors

  • Monitor markers of active translation (e.g., puromycin incorporation, ribosome profiling)

  • Correlate with expression of known EIF4H-dependent proteins (FGF-2, VEGF, c-Myc, cyclin D1)

• Technical challenges and solutions:

  • Tissue heterogeneity requires careful region selection for analysis

  • Standardized sampling approaches with grid-based assessment

  • Automated quantification algorithms reduce observer bias

  • Serial sections for complementary assays ensure data integration

How can researchers troubleshoot non-specific signals in EIF4H western blotting applications?

When encountering non-specific bands or background with EIF4H antibodies in western blotting:

• Blocking optimization:

  • Test alternative blocking agents (BSA vs. non-fat milk)

  • Increase blocking time to 2 hours at room temperature

  • Consider specialized blocking reagents for polyclonal antibodies

• Antibody dilution and incubation parameters:

  • Titrate antibody concentration within validated range (0.04-0.4 μg/mL)

  • Extended incubation at 4°C overnight may improve signal-to-noise ratio

  • Validate optimal dilution for each new antibody lot

• Addressing cross-reactivity:

  • Perform parallel detection in EIF4H knockdown samples as negative controls

  • Consider pre-adsorption with recombinant protein for highly cross-reactive antibodies

  • Validate with antibodies targeting different epitopes to confirm band identity

• Gel separation considerations:

  • Use higher percentage gels (12-15%) to better resolve the closely sized 25 kDa and 27 kDa isoforms

  • Extend running time to improve separation of similar molecular weight proteins

  • Consider gradient gels for simultaneous detection of EIF4H and interaction partners

What strategies can improve detection sensitivity in EIF4H immunohistochemistry of archived tissue samples?

Working with archived or challenging tissue samples requires specialized approaches:

• Enhanced antigen retrieval methods:

  • Extended heat-induced epitope retrieval (20-40 minutes)

  • Trial of alternative pH buffers (citrate pH 6.0 vs. EDTA pH 9.0)

  • Enzymatic retrieval as an alternative for certain fixation conditions

• Signal amplification options:

  • Tyramide signal amplification can significantly enhance detection sensitivity

  • Polymer-based detection systems often outperform avidin-biotin methods

  • Consider multimer-based detection technologies for low-abundance targets

• Background reduction strategies:

  • Implement dual endogenous blocking (peroxidase and alkaline phosphatase)

  • Include avidin-biotin blocking step regardless of detection system

  • Consider specialized blocking for tissue-specific elements (melanin, hemosiderin)

• Optimization for different tissue types:

  • Lung tissue may require specialized deparaffinization due to airspace artifacts

  • Brain tissue often benefits from extended permeabilization steps

  • Bone marrow samples may require decalcification-compatible antibody clones

How can researchers quantitatively assess EIF4H expression levels across different experimental systems?

Standardized quantification approaches are essential for comparative EIF4H analysis:

• Western blot quantification strategies:

  • Implement loading controls appropriate for your experimental context

  • Consider normalization to total protein stains rather than housekeeping proteins

  • Use calibrated recombinant standards for absolute quantification

• Immunohistochemistry quantification:

  • Employ digital pathology systems with validated algorithms

  • Develop H-score or Allred-type scoring systems incorporating intensity and percentage

  • Include reference standards in each staining batch

• Flow cytometry applications:

  • Optimize fixation and permeabilization for intracellular EIF4H detection

  • Use median fluorescence intensity for quantitative comparisons

  • Include calibration beads to standardize across experiments

• Cross-platform normalization approaches:

  • Establish reference standards measured across all platforms

  • Develop conversion factors between different quantification methods

  • Consider orthogonal validation with mass spectrometry-based proteomics