EIF6 Antibody

What is the subcellular localization of EIF6 and how does this impact antibody selection?

EIF6 exhibits dual localization in both the nucleus and cytoplasm, which has important implications for antibody-based detection strategies. The protein features up to two different isoforms that must be considered when selecting detection reagents . When choosing an appropriate antibody, researchers should consider whether their experimental goals require detection of EIF6 in specific subcellular compartments.

For optimal results in subcellular localization studies, immunofluorescence (IF) or immunocytochemistry (ICC) applications are recommended with antibodies validated for these specific techniques. Several commercially available antibodies have been specifically validated for detecting the nuclear and cytoplasmic pools of EIF6, with those targeting amino acids 66-210 showing reliable performance across multiple species including human, mouse, and rat samples .

What expression patterns of EIF6 should researchers expect across different tissue types?

EIF6 exhibits a distinctive tissue-specific expression pattern that researchers should account for when designing experiments. The protein is expressed at very high levels in colon carcinoma with comparatively lower levels in normal colon and ileum tissues, and lowest expression in kidney and muscle . This differential expression pattern makes EIF6 particularly relevant for cancer research applications.

When conducting comparative tissue studies, researchers should calibrate antibody dilutions according to the expected expression levels. Western blot protocols may require optimization with longer exposure times for low-expressing tissues like kidney and muscle, while immunohistochemistry applications might benefit from signal amplification techniques in these tissues. Researchers should validate their antibody using positive control samples (colon carcinoma) alongside experimental tissues to ensure detection sensitivity across the expression spectrum .

What are the most validated applications for EIF6 antibodies, and what controls should be implemented?

The most extensively validated applications for EIF6 antibodies include Western Blot (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF). Based on citation frequency and commercial validation, Western Blot appears to be the most widely utilized application . For comprehensive experimental design, appropriate controls are essential.

When implementing EIF6 antibody-based techniques, researchers should include:

• Positive controls: Colon carcinoma tissue/cell lines with known high EIF6 expression

• Negative controls: Primary antibody omission

• Specificity controls: Blocking peptide competition assays

• Loading controls: For WB applications, proteins such as β-actin or GAPDH

• Cross-reactivity assessment: Particularly when working with non-human models, as EIF6 shares 99.3% amino acid sequence identity between human, mouse, and rat

For optimal sensitivity and specificity, antibodies targeting amino acid regions 66-210 have demonstrated reliable performance across multiple applications and species .

How can researchers investigate EIF6-60S interactions using antibody-based techniques?

The interaction between EIF6 and the 60S ribosomal subunit represents a critical regulatory node in translation initiation. To investigate this interaction, researchers can employ sophisticated antibody-based approaches that go beyond simple detection.

Co-immunoprecipitation (Co-IP) using anti-EIF6 antibodies allows isolation of EIF6-60S complexes from cellular lysates. For optimal results, use affinity-purified antibodies targeting regions away from the 60S binding interface (avoid Y151 region) to prevent interference with the interaction . Implement the following protocol optimization steps:

• Use mild lysis conditions (e.g., 50 mM Tris-HCl, pH 8.0, 300 mM NaCl) to preserve native protein complexes

• Add RNase inhibitors to protect rRNA integrity within the 60S subunit

• Consider crosslinking approaches for transient interactions

• Validate interactions through reciprocal Co-IP using antibodies against uL14 (RPL23)

Proximity ligation assays (PLA) offer an alternative approach to visualize EIF6-60S interactions in situ with subcellular resolution. This technique requires antibodies raised in different host species against EIF6 and ribosomal proteins like uL14. Recent research has shown that disrupting key residues in the EIF6-60S binding interface, particularly at the Y151 position, markedly affects cancer cell proliferation, highlighting a potential therapeutic target .

What methodological approaches can detect dynamic conformational changes in EIF6 upon binding to uL14?

Recent research has revealed that EIF6 undergoes dynamic conformational changes upon binding to uL14, suggesting an allosteric regulatory mechanism. While hydrogen-deuterium exchange mass spectrometry (HDX-MS) has been instrumental in uncovering these changes , researchers can employ antibody-based methods to further investigate these dynamics.

A multi-faceted approach combining conformation-specific antibodies with functional assays is recommended:

• Epitope mapping with antibody panels: Utilize multiple antibodies targeting different EIF6 epitopes to detect conformational shifts. Reduced binding of certain antibodies after uL14 interaction may indicate conformational masking of epitopes.

• FRET-based assays: Implement FRET (Förster Resonance Energy Transfer) using fluorescently labeled antibodies against EIF6 and uL14 to detect proximity and conformational changes in real-time.

• Phosphorylation-specific antibodies: Since phosphorylation of S235 in the C-tail has been implicated in EIF6 release from 60S , phospho-specific antibodies can monitor this regulatory mechanism.

For researchers investigating the allosteric interface regulated by the C-tail of EIF6, circular dichroism (CD) studies have shown that negative charges at conserved phosphorylation sites in the C-tail significantly influence EIF6 conformation . This approach can be complemented with antibodies that specifically recognize these phosphorylated states.

How can researchers experimentally investigate EIF6 rebinding to post-termination 60S subunits?

The dynamic recycling of EIF6, particularly its rebinding to post-termination 60S ribosomal subunits, represents an emerging area of interest in translation regulation. Researchers can establish an ex vivo assay coupling EIF6 release from 60S subunits to their reassembly into 80S ribosomes.

The following protocol framework can be implemented:

• Isolate eIF6-loaded 60S subunits from c-kit+ bone marrow cells

• Add recombinant human SBDS, EFL1, and GTP to promote EIF6 release

• Monitor EIF6 redistribution via sucrose gradient fractionation followed by immunoblotting with anti-EIF6 antibodies

• Quantify 80S ribosome reassembly as a functional readout

This assay has demonstrated that SBDS and EFL1 are the minimal components required to recycle 60S subunits by evicting EIF6, with a measurable 1.8-fold increase in 80S ribosome reassembly upon successful treatment . For in vivo validation, researchers can use doxycycline-inducible transgenic models that permit graded overexpression of EIF6, thereby allowing investigation of dose-dependent effects on ribosomal subunit joining .

What are the common challenges in obtaining soluble recombinant EIF6 for antibody validation, and how can they be overcome?

A significant challenge in EIF6 research has been obtaining sufficient quantities of active full-length human EIF6 for antibody validation and functional studies. The search results indicate specific approaches to address this challenge.

Researchers can implement co-expression of molecular chaperones to enhance EIF6 solubility:

• Co-transform expression vectors with chaperone-expressing plasmids such as pTf16 (for trigger factor chaperone)

• Culture in LB media supplemented with appropriate antibiotics (kanamycin 50 μg/ml, chloramphenicol 34 μg/ml)

• Add L-arabinose (2 mg/ml) to induce chaperone expression

• Induce EIF6 expression with 0.4 mM IPTG at OD600 0.4-0.5

• Extend expression at reduced temperature (20°C for 20 hours)

• Use lysis buffer containing 50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM β-mercaptoethanol, and 1 mM PMSF

For optimal purification after co-expression with chaperones, include size-exclusion chromatography on a Superdex S200 column to remove all chaperones prior to antibody validation steps . This approach has successfully yielded milligram quantities of active full-length EIF6, enabling detailed biophysical characterization and proper antibody validation.

What strategies can address non-specific binding issues with EIF6 antibodies in complex tissue samples?

When working with EIF6 antibodies in complex tissue samples, researchers may encounter non-specific binding that complicates data interpretation. Several methodological approaches can minimize these issues:

• Antibody selection: Choose antibodies with demonstrated specificity. For example, those targeting amino acids 66-210 have shown reliable specificity across human, mouse, and rat samples with minimal cross-reactivity .

• Blocking optimization: Implement a dual blocking approach using both:

  • 5% BSA in TBS-T for general blocking

  • 2% normal serum from the same species as the secondary antibody

• Antigen retrieval calibration: For IHC applications, optimize antigen retrieval conditions:

  • Test both citrate buffer (pH 6.0) and EDTA buffer (pH 9.0)

  • Adjust heating time incrementally (10-20 minutes)

• Validation controls:

  • Include peptide competition assays using the specific immunogen (EIF6 amino acids 66-210)

  • Run parallel staining with multiple antibodies targeting different EIF6 epitopes

  • Include known negative tissues (based on transcriptomic data)

• Signal amplification alternatives: When working with tissues known to have low EIF6 expression (kidney, muscle), consider:

  • Tyramide signal amplification for IHC/IF

  • Enhanced chemiluminescence reagents for Western blot

By implementing these methodological refinements, researchers can significantly improve signal-to-noise ratios and ensure reliable detection of EIF6 across diverse experimental contexts.

How can EIF6 antibodies be utilized to investigate its role in cancer research?

EIF6 exhibits notably elevated expression in colon carcinoma compared to normal tissues , suggesting a potential role in cancer pathophysiology. Researchers investigating EIF6 in oncology can implement several antibody-based approaches:

• Comparative expression profiling: Use validated antibodies for IHC to quantify EIF6 expression across tumor grades, stages, and subtypes. Establish standardized scoring systems (H-score or Allred) for consistent quantification.

• Targeting the EIF6-60S interface: Recent research has shown that disrupting key residues in the EIF6-60S binding interface, particularly Y151, markedly limits cancer cell proliferation . Researchers can:

  • Develop phospho-specific antibodies targeting Y151 modifications

  • Utilize antibodies in high-content screening for compounds disrupting this interaction

  • Monitor treatment response through EIF6-60S dissociation in patient-derived xenografts

• Translational activity correlation: Combine anti-EIF6 immunoprecipitation with polysome profiling to assess the relationship between EIF6 levels and global translation rates in tumor versus normal tissues.

• Post-translational modification mapping: Since phosphorylation at S235 appears to regulate EIF6 function , phospho-specific antibodies can help characterize the activation state of EIF6 in different cancer types and stages.

This multi-faceted approach can provide insights into how EIF6 dysregulation contributes to cancer pathogenesis and identify potential therapeutic vulnerabilities by targeting its interaction with the 60S ribosomal subunit.

What methodological approaches can detect EIF6 involvement in Shwachman-Diamond syndrome?

Shwachman-Diamond syndrome (SDS) is a leukemia predisposition disorder associated with mutations in SBDS and EFL1, factors that release EIF6 from 60S ribosomal subunits . Researchers investigating this rare disease can implement several antibody-based methodological approaches:

• Ex vivo ribosomal recycling assays: Establish a biochemical assay using:

  • EIF6-loaded 60S subunits from patient-derived cells

  • Recombinant wild-type or mutant SBDS and EFL1 proteins

  • Detection of EIF6 release via immunoblotting

  • Quantification of 80S ribosome reassembly as functional readout

• Hematopoietic differentiation analysis: For erythropoiesis studies in SDS, monitor:

  • EIF6 levels throughout differentiation using flow cytometry with anti-EIF6 antibodies

  • Polysome profiles with quantification of free 60S subunits

  • Global protein synthesis rates correlated with EIF6 sequestration patterns

• Therapeutic response monitoring: When testing potential SDS therapeutics, use anti-EIF6 antibodies to:

  • Measure restoration of normal EIF6 localization patterns

  • Quantify improvements in 60S subunit availability

  • Assess normalization of ribosomal subunit joining rates

The search results indicate that increased EIF6 levels impair terminal erythropoiesis by sequestering post-termination 60S subunits in the cytoplasm, disrupting subunit joining and attenuating global protein synthesis . This mechanistic insight provides a rational basis for therapeutic approaches targeting the EIF6-60S interaction in SDS patients.

How can researchers investigate the allosteric regulation of EIF6 using antibody-based methods?

Recent research has uncovered an allosteric interface in EIF6 regulated by its C-tail, with phosphorylation at S235 proposed to release EIF6 from the 60S subunit . Researchers can implement several antibody-based approaches to investigate this regulatory mechanism:

• Conformation-specific antibodies: Develop antibodies that specifically recognize the "open" versus "closed" conformational states of EIF6, particularly focusing on regions that undergo structural changes upon C-tail phosphorylation.

• Phospho-specific antibody applications: Utilize antibodies that specifically recognize phosphorylated S235 to:

  • Quantify the proportion of phosphorylated EIF6 in different cellular compartments

  • Identify kinases responsible for this modification through co-immunoprecipitation

  • Monitor temporal dynamics of phosphorylation during translation cycles

• Structure-function correlation: Combine site-directed mutagenesis of key residues (identified through circular dichroism studies) with antibody-based detection to establish structure-function relationships.

This methodological approach will allow researchers to dissect the molecular mechanisms underlying allosteric regulation of EIF6 and potentially identify new therapeutic targets for diseases characterized by dysregulated translation.

What experimental design considerations are important when using EIF6 antibodies for advanced ribosome profiling studies?

Ribosome profiling provides genome-wide information about translation, and incorporating EIF6 analysis can yield insights into translation regulation mechanisms. Researchers should consider the following methodological approach:

• Antibody-based ribosome isolation: Use anti-EIF6 antibodies to specifically isolate EIF6-bound 60S subunits prior to ribosome profiling, allowing characterization of:

  • mRNAs specifically associated with EIF6-bound versus EIF6-free 60S subunits

  • Differential translation efficiency of specific transcripts under varying EIF6 conditions

  • Potential specialized functions of EIF6-bound ribosomes

• Combined approaches: Integrate antibody-based techniques with ribosome profiling by:

  • Performing sequential immunoprecipitation of EIF6 followed by ribosome footprinting

  • Correlating EIF6 phosphorylation status with ribosome positioning on mRNAs

  • Comparing translational landscapes between wild-type and EIF6 mutant conditions

• Validation controls: Implement appropriate controls including:

  • Parallel profiling with isotype control antibodies

  • Spike-in standards for normalization

  • Profile comparison between EIF6 knockdown/overexpression conditions

This integrated approach can provide unprecedented insights into how EIF6 influences the translational landscape and identify specific mRNA targets or pathways that are differentially regulated through EIF6-dependent mechanisms.