EIF1AD Antibody

What is EIF1AD and why is it a target of interest in research?

EIF1AD (Eukaryotic Translation Initiation Factor 1A Domain Containing) is a protein involved in cellular response to oxidative stress and plays a role in decreasing cell proliferation . It contains domains similar to translation initiation factors and has been implicated in translational initiation processes . Research interest in EIF1AD has increased following its identification as a potential regulator of tau pathology in neurodegenerative diseases, particularly Alzheimer's disease .

What applications are EIF1AD antibodies typically validated for?

EIF1AD antibodies are validated for multiple research applications including:

Research applications should incorporate positive controls and proper validation to ensure specificity of antibody binding .

How should EIF1AD antibodies be stored and handled to maintain optimal activity?

Most EIF1AD antibodies should be stored at -20°C and are typically supplied in PBS with sodium azide and glycerol (often 50% glycerol, pH 7.3) . Critical storage recommendations include:

• Avoid repeated freeze-thaw cycles

• Some products specifically indicate "DO NOT ALIQUOT"

• For working solutions, store at 2-8°C for short term (typically up to one month)

• Most products maintain stability for 6-12 months at -20°C to -70°C under sterile conditions after reconstitution

Note that improper storage can significantly impact antibody performance in downstream applications.

What factors should be considered when selecting between polyclonal and monoclonal EIF1AD antibodies?

The choice between polyclonal and monoclonal EIF1AD antibodies depends on research objectives:

Most commercially available EIF1AD antibodies appear to be rabbit polyclonal antibodies , which provide robust detection across multiple applications but may require additional validation for highly specific applications.

What is the optimal methodology for validating EIF1AD antibody specificity?

A comprehensive validation strategy should include:

• Western blot analysis: Confirm correct molecular weight (~27 kDa for human EIF1AD)

• Positive control samples: Use validated cell lines (K-562, HEK-293, A549, HL-60, MCF7)

• Negative controls: Include antibody diluent without primary antibody

• Knockdown/knockout validation: Compare signal in normal vs. EIF1AD-depleted samples using CRISPR/Cas9 or siRNA approaches

• Cross-reactivity assessment: Test antibody against related proteins, particularly other translation initiation factors

• Species reactivity confirmation: Verify expected patterns in human vs. mouse/rat samples if using across species

Most commercial antibodies undergo some validation procedures, but researchers should conduct application-specific validation in their experimental systems.

What dilution ranges are recommended for different applications of EIF1AD antibodies?

Optimal dilution ranges vary by application and product:

Note that these ranges serve as starting points and should be optimized for each specific antibody and experimental condition .

How can EIF1AD antibodies be effectively employed in studies of protein-protein interactions and cellular localization?

For protein-protein interaction and localization studies:

• Co-immunoprecipitation (Co-IP):

  • Use EIF1AD antibodies conjugated to agarose/magnetic beads

  • Ensure antibody does not interfere with protein interaction domains

  • Include proper controls (IgG control, reverse Co-IP)

  • Validate interactions using additional methods (proximity ligation assay, FRET)

• Subcellular localization:

  • Immunofluorescence data suggests EIF1AD localizes to intermediate filament cytoskeleton and nucleoplasm

  • Use counterstains for specific organelles (DAPI for nucleus, MitoTracker for mitochondria)

  • Confirm findings with cell fractionation followed by Western blot

  • Consider both fixation-dependent artifacts and potential translocation under different cellular conditions

Research has demonstrated the importance of proper methodology when studying EIF1AD interactions with translation machinery components and stress response pathways.

What considerations are important when using EIF1AD antibodies in neurodegenerative disease research?

Recent research has identified EIF1AD as a regulator of tau pathology , making it relevant for neurodegenerative disease studies:

• Technical considerations:

  • Use antibodies validated in neuronal tissues/cell lines

  • Employ multiple detection methods (IHC, WB, IF) for comprehensive analysis

  • Consider post-translational modifications that may affect antibody recognition

• Experimental approaches:

  • Combine with CRISPR-based approaches to examine functional consequences of EIF1AD modulation

  • Use sarkosyl fractionation to separate soluble and insoluble tau species when studying EIF1AD's role in tau aggregation

  • Monitor tau aggregation in biosensor cells after EIF1AD knockdown using FRET-based assays

• Clinical relevance:

  • EIF1AD is downregulated in Alzheimer's disease brains, suggesting its decreased activity may contribute to tau pathology progression

  • Multiple isoforms of EIF1AD (including a 19 kD canonical form) have been detected in human brain tissue

How can EIF1AD antibodies be integrated with CRISPR/Cas9 technologies in functional genomic studies?

Integration of EIF1AD antibodies with CRISPR technologies offers powerful research approaches:

• Antibody validation using CRISPR-generated knockouts:

  • Generate EIF1AD knockout cell lines using available sgRNA CRISPR/Cas9 All-in-One vector systems

  • Use antibodies to confirm knockout efficiency via Western blotting

  • Test for antibody specificity by confirming loss of signal in knockout cells

• Functional genomics applications:

  • Utilize EIF1AD sgRNA CRISPR/Cas9 systems to create knockdowns/knockouts

  • Employ antibodies to:

    • Confirm target depletion

    • Assess effects on interacting partners

    • Determine consequences for downstream cellular processes

  • Combine with phenotypic assays (e.g., tau aggregation) to correlate EIF1AD levels with functional outcomes

• Recommended workflow for CRISPR-antibody studies:

  • Generate stable cell lines expressing Cas9 and sgRNAs targeting EIF1AD

  • Confirm knockdown/knockout efficiency using validated EIF1AD antibodies

  • Analyze phenotypic consequences (cellular function, protein-protein interactions)

  • Perform rescue experiments with sgRNA-resistant EIF1AD constructs

What are common issues encountered with EIF1AD antibodies and how can they be resolved?

How should researchers approach conflicting results between different EIF1AD antibodies or detection methods?

When confronted with conflicting results:

• Antibody validation assessment:

  • Evaluate how thoroughly each antibody was validated

  • Check if the antibodies target different epitopes

  • Review published literature for similar discrepancies

• Technical validation:

  • Use orthogonal approaches (e.g., mass spectrometry) to confirm findings

  • Test multiple antibodies targeting different regions of EIF1AD

  • Implement genetic approaches (CRISPR/RNAi) to validate specificity

• Biological considerations:

  • Determine if discrepancies reflect biological variables (cell type, treatment conditions)

  • Consider post-translational modifications that might affect epitope recognition

  • Evaluate potential alternative splice variants or protein isoforms

• Methodological approach:

  • Document all experimental conditions thoroughly

  • Perform side-by-side comparisons using standardized samples

  • Consider whether different applications (WB vs. IHC) might yield different results due to protein conformation differences

What critical considerations should be addressed when analyzing EIF1AD expression levels in disease states?

When studying EIF1AD in disease contexts (particularly neurodegenerative diseases ):

• Sample selection and preparation:

  • Match cases and controls for age, sex, post-mortem interval

  • Use consistent collection, storage, and extraction protocols

  • Consider regional variation in brain samples for neurodegenerative studies

• Technical controls:

  • Include loading controls appropriate for the tissue/condition studied

  • Use multiple reference genes for normalization in qPCR studies

  • Consider both protein and mRNA levels to identify post-transcriptional regulation

• Data interpretation:

  • Correlate EIF1AD levels with disease markers (e.g., tau pathology measures)

  • Account for potential confounding factors (medication, comorbidities)

  • Consider cell-type specific changes versus bulk tissue measurements

  • Recent studies found EIF1AD downregulation in Alzheimer's disease brains, suggesting this may contribute to tau pathology progression

• Validation approaches:

  • Confirm findings in independent cohorts

  • Use multiple antibodies targeting different epitopes

  • Validate with complementary techniques (IHC, WB, qPCR)

How is EIF1AD related to tau pathology and what methodological approaches can investigate this connection?

Recent genome-wide CRISPRi screening identified EIF1AD as a regulator of tau pathology , with the following key findings and methodological approaches:

• Functional genomic screening:

  • CRISPR-based screening in tau biosensor cells identified EIF1AD knockdown as promoting tau aggregation

  • Methodology involved tau biosensor cells expressing FRET pair (tau RD-CFP and tau RD-YFP) with KRAB-dCas9

  • Cells were sorted into FRET(+) and FRET(-) populations to identify genes affecting tau aggregation

• Validation methodology:

  • Individual sgRNAs silencing EIF1AD confirmed increased tau aggregation

  • Tested with both exosomal and vesicle-free tau seeds

  • Quantification via FRET flow cytometry

• Clinical correlation:

  • Western blot analysis showed decreased EIF1AD levels in Alzheimer's disease brains

  • Both canonical (19 kDa) and lower molecular weight variants were detected

  • Suggests that EIF1AD downregulation may contribute to tau pathology progression in human brain

• Mechanistic investigation approaches:

  • EIF1AD is implicated in final steps of 40S ribosomal subunit maturation

  • Methodologies to explore mechanistic connections include ribosome profiling, translational efficiency assays, and protein synthesis measurements

What novel applications of EIF1AD antibodies are emerging in current research?

Emerging applications for EIF1AD antibodies include:

• Biomarker development:

  • Potential use as prognostic/diagnostic markers in neurodegenerative diseases

  • Methodological approaches include examining EIF1AD levels in:

    • Post-mortem brain tissue from different disease stages

    • CSF and blood samples from patients vs. controls

    • Correlation with disease progression measures

• Therapeutic target validation:

  • Using antibodies to track EIF1AD modulation by candidate drugs

  • Monitoring effects of EIF1AD restoration on tau pathology

  • High-throughput screening for compounds that normalize EIF1AD levels

• Single-cell applications:

  • Integration with single-cell technologies to examine cell-type specific expression

  • Methods include single-cell Western blotting, mass cytometry, and imaging cytometry

  • Correlation with cellular vulnerability in neurodegenerative diseases

• Structure-function studies:

  • Using domain-specific antibodies to investigate functional regions

  • Examining post-translational modifications that affect activity

  • Probing conformational changes under different cellular conditions

What is the relationship between EIF1AD and cellular stress responses in disease models?

EIF1AD plays a role in cellular response to oxidative stress , with implications for disease research:

• Methodological approaches to study stress relationships:

  • Examine EIF1AD expression/localization changes under various stressors:

    • Oxidative stress (H₂O₂, paraquat)

    • ER stress (tunicamycin, thapsigargin)

    • Proteotoxic stress (proteasome inhibitors)

  • Use time-course studies to determine acute vs. chronic responses

  • Combine with phospho-specific antibodies to track stress-induced modifications

• Functional investigation methods:

  • Compare stress vulnerability in EIF1AD knockdown/knockout models

  • Assess impact on integrated stress response markers (eIF2α phosphorylation, ATF4 induction)

  • Measure translational efficiency during stress recovery

• Disease relevance:

  • In neurodegenerative disease models, EIF1AD levels correlate with susceptibility to tau pathology

  • Methodologies to investigate this include:

    • Stress-induction in tau biosensor cells with modulated EIF1AD levels

    • Examination of stress markers in brain regions with varying tau pathology

    • Correlation of oxidative damage markers with EIF1AD and tau in patient samples