eif3jb Antibody

What is eIF3jb and how does it differ from other eIF3 subunits?

eIF3jb (eukaryotic translation initiation factor 3, subunit Jb) is a protein found in zebrafish (Danio rerio) that belongs to the eukaryotic translation initiation factor family. It differs from other eIF3 subunits in several important ways:

• While most eIF3 subunits are stable components of the eIF3 complex, evidence suggests eIF3j (the mammalian counterpart of zebrafish eIF3jb) often functions in an eIF3-independent manner and is not considered a bona fide eIF3 subunit .

• Unlike core eIF3 subunits like eIF3a and eIF3b that form the nucleation core of the complex, eIF3j has been identified as a regulatory component that can actually inhibit translation of certain RNAs .

• eIF3jb in zebrafish is expressed in specific structures including lens, midbrain, musculature system, polster, and pronephric duct during development .

• The zebrafish eIF3jb protein contains an eukaryotic translation initiation factor 3-like domain and is 263 amino acids in length .

When selecting antibodies for research, it's crucial to understand these distinctions, particularly if comparing results across species or investigating translation regulation mechanisms.

What experimental applications are eIF3jb antibodies typically used for?

Based on data from related eIF3j antibodies, eIF3jb antibodies are suitable for multiple research applications:

When working with zebrafish-specific eIF3jb antibodies, validation is essential as reactivity may differ from human/mouse EIF3J antibodies. Cross-reactivity testing is recommended before proceeding with full experiments .

How should I design proper controls for eIF3jb antibody experiments in zebrafish models?

Designing robust controls for eIF3jb antibody experiments requires multiple layers of validation:

Essential controls for zebrafish eIF3jb antibody experiments:

• Specificity controls:

  • Knockdown validation: Use morpholinos or CRISPR to reduce eIF3jb expression and confirm reduced antibody signal

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide to block specific binding

  • Cross-reactivity assessment: Test antibody against recombinant eIF3jb and related proteins (eIF3ja, human EIF3J) to confirm specificity

• Technical controls:

  • Negative controls: Primary antibody omission and isotype controls to assess background

  • Positive controls: Use tissues with known eIF3jb expression (lens, midbrain) based on in situ hybridization data

  • Loading controls: For western blots, use stable reference proteins (α-Tubulin, HDAC1)

• Experimental controls:

  • Wild-type vs. mutant comparison

  • Developmental stage controls (expression varies by stage)

  • Tissue-specific controls based on known expression patterns in lens, midbrain, etc.

Remember that endogenous controls also help assess cell/tissue health and can indicate whether experimental issues stem from the protocol or sample preparation11.

What are the key differences in experimental approach when using eIF3jb antibodies for studying translation versus protein localization?

The experimental approaches differ substantially based on your research question:

For translation studies:

• Focus on cell fractionation to isolate ribosomal complexes

• Consider using RNA-immunoprecipitation (RIP) to identify mRNAs associated with eIF3jb

• Include translation inhibitors (cycloheximide, puromycin) as controls

• Measure protein synthesis rates with metabolic labeling (35S-methionine)

• Design experiments to capture the inhibitory effect of eIF3j on circular RNA translation

For protein localization studies:

• Optimize fixation protocols (4% paraformaldehyde is standard for zebrafish)

• Use confocal microscopy with Z-stack imaging for tissue analysis

• Include co-staining with known markers (nuclear, ribosomal, etc.)

• Consider both fluorescent and chromogenic detection methods

• Validate subcellular localization with fractionation followed by western blotting

For both approaches, consider that eIF3jb has been shown to associate with the aminoacyl site and mRNA entry channel of the 40S ribosomal subunit and plays a role in recycling post-termination complexes . This suggests examining both nuclear and cytoplasmic fractions in your experiments.

How can I use eIF3jb antibodies to investigate its role in circular RNA translation regulation?

Recent research has identified eIF3j as a potent inhibitor of circular RNA translation . To investigate this function of eIF3jb in zebrafish:

• Experimental design approach:

  • Establish a reporter system using known translatable circular RNAs

  • Create constructs with wild-type and mutant circular RNA untranslated regions (UTRs)

  • Implement heat shock experiments to test stress response regulation

• Methodological workflow:

  • Use immunoprecipitation with eIF3jb antibodies to capture associated circular RNAs

  • Perform RNA-seq on the immunoprecipitated material to identify bound circular RNAs

  • Validate binding using RNA electrophoretic mobility shift assays (EMSA)

  • Conduct translation assays in the presence/absence of eIF3jb

• Critical controls:

  • Compare circular RNA translation efficiency with and without eIF3jb knockdown

  • Test the C-terminus of eIF3jb specifically, as this region is essential for its inhibitory effect

  • Include linear mRNA controls to distinguish circular RNA-specific effects

The research indicates that eIF3j binds to an RNA regulon within circular RNA UTRs to promote dissociation of the eIF3 complex, thereby inhibiting translation . Your antibody-based approach should focus on capturing these interactions and measuring the resulting translation efficiency changes.

What are the most effective protocols for using eIF3jb antibodies in immunoprecipitation studies examining protein-protein interactions in the translation initiation complex?

For robust immunoprecipitation (IP) studies of eIF3jb interactions:

Optimized IP Protocol:

• Lysis buffer selection:

  • Use a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 2 mM EDTA with protease inhibitors

  • For studying RNA-dependent interactions, include RNase inhibitors

  • For capturing weak interactions, consider crosslinking with formaldehyde (0.1-0.3%)

• Antibody amounts and conditions:

  • Use 0.5-4.0 μg antibody per 1-3 mg of total protein lysate

  • Pre-clear lysate with Protein A/G beads for 1 hour at 4°C

  • Incubate with antibody overnight at 4°C with gentle rotation

• Detection strategies:

  • Western blotting: Use 1:1000 dilution for most eIF3 subunit antibodies

  • Mass spectrometry: For unbiased identification of interacting partners

  • RNA analysis: Include RT-PCR to identify associated RNAs

Control recommendations:

• IgG control antibody of the same species and isotype

• Input samples (typically 5-10% of IP material)

• Reciprocal IPs with antibodies against known interaction partners

• RNase treatment controls to distinguish RNA-dependent interactions

This approach has successfully identified interactions between eIF3j and components of the 40S ribosomal subunit as well as its role in post-termination complex recycling .

How do I address false positive results when using eIF3jb antibodies in zebrafish tissue immunohistochemistry?

False positives are a common challenge when using antibodies in zebrafish. Address them systematically:

• Sources of false positives:

  • Cross-reactivity with related proteins (eIF3ja or other eIF3 subunits)

  • Non-specific binding to highly abundant proteins

  • Inadequate blocking or excessive antibody concentration

  • Autofluorescence from zebrafish tissues (particularly yolk)

• Validation strategies:

  • Peptide competition: Pre-incubate antibody with immunizing peptide

  • Knockout/knockdown controls: Use morpholinos or CRISPR-generated eIF3jb mutants

  • Multiple antibody approach: Use antibodies targeting different epitopes

  • Correlate with mRNA expression: Compare with in situ hybridization patterns

• Protocol optimization:

  • Titrate antibody concentration (start at 1:20 and adjust as needed)

  • Test multiple antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Increase blocking time/concentration (5% BSA or 10% normal serum)

  • Include detergents to reduce background (0.1-0.3% Triton X-100)

• Alternative approaches:

  • Consider using tagged versions of eIF3jb for cleaner detection

  • Employ tyramide signal amplification for weak signals while maintaining specificity

  • Use fluorescent in situ hybridization (FISH) as a complementary approach

Remember that zebrafish-specific antibodies may be limited, so careful validation is essential when using antibodies developed against human or mouse orthologs.

What are the most common pitfalls when interpreting western blot results for eIF3jb and how can I ensure accurate molecular weight detection?

Interpreting eIF3jb western blots requires careful attention to several technical considerations:

Remember that the observed molecular weight of 35 kDa for human EIF3J may differ slightly for zebrafish eIF3jb due to species differences in protein sequence and modifications.

How can I use eIF3jb antibodies to investigate stress-responsive translation regulation mechanisms in zebrafish models?

Recent research has revealed that eIF3j plays a role in stress-responsive translation regulation, particularly during heat shock . To investigate this in zebrafish:

• Experimental design:

  • Subject zebrafish to controlled stress conditions (heat shock, hypoxia, etc.)

  • Collect tissues at various timepoints post-stress

  • Fractionate cells to separate actively translating ribosomes

  • Use eIF3jb antibodies to track localization and interaction changes

• Specific methodologies:

  • Polysome profiling with eIF3jb immunoblotting of fractions

  • Proximity ligation assay (PLA) to detect stress-induced changes in eIF3jb-protein interactions

  • Ribosome footprinting combined with eIF3jb immunoprecipitation

  • Live imaging with fluorescently tagged eIF3jb to track dynamic responses

• Key measurements:

  • Changes in eIF3jb association with circular RNAs during stress

  • Alterations in eIF3jb phosphorylation state (using phospho-specific antibodies)

  • Shifts in eIF3jb subcellular localization

  • Changes in translation rates of specific mRNAs

This approach leverages the finding that eIF3j regulates heat resistance by attenuating translation of certain circular RNAs during heat stress , which may be similarly conserved in zebrafish eIF3jb.

What are the best practices for using eIF3jb antibodies in multi-color flow cytometry experiments examining translation initiation complex formation?

For multi-color flow cytometry experiments investigating translation initiation complexes:

• Panel design considerations:

  • Include markers for cell cycle phases (DNA content staining)

  • Add antibodies against other eIF3 subunits (particularly eIF3a and eIF3b as core components)

  • Consider markers for stress response and cell viability

  • Example panel design:

• Sample preparation optimization:

  • Use gentle fixation (2% paraformaldehyde, 10 minutes)

  • Permeabilize with 0.1% Triton X-100 or saponin buffer

  • Include RNase inhibitors if RNA-protein interactions are important

  • Block with 2-5% BSA to reduce background

• Critical controls:

  • Single-color compensation controls for each fluorochrome

  • Fluorescence Minus One (FMO) controls to set proper gates

  • Isotype controls matched to antibody class and fluorophore

  • Biological controls (untreated vs. treated cells)

• Analysis considerations:

  • Gate on single, viable cells first

  • Analyze eIF3jb levels in relation to cell cycle phases

  • Look for co-expression patterns with other eIF3 subunits

  • Consider using dimension reduction techniques (tSNE, UMAP) for complex datasets

This approach allows for quantitative assessment of eIF3jb expression and its correlation with translation complex formation at the single-cell level .

How can eIF3jb antibodies be utilized to study the role of translation dysregulation in zebrafish cancer models?

Translation dysregulation is a hallmark of cancer, and eIF3 subunits have been implicated in human cancer progression . To investigate eIF3jb's role in zebrafish cancer models:

• Experimental model systems:

  • Transgenic zebrafish expressing oncogenes (KRAS^G12D^, BRAF^V600E^)

  • Chemically-induced cancer models (DMBA, ethylnitrosourea)

  • Xenograft models with fluorescently labeled human cancer cells

  • CRISPR-engineered eIF3jb mutant lines

• Analytical approaches:

  • Immunohistochemistry to assess eIF3jb expression in tumor vs. normal tissue

  • Western blotting to quantify expression changes during cancer progression

  • RNA-immunoprecipitation to identify cancer-specific mRNAs regulated by eIF3jb

  • Polysome profiling to assess global translation changes

• Research questions to address:

  • Does eIF3jb expression correlate with tumor grade/progression as observed for eIF3b in human bladder and prostate cancer?

  • What circular RNAs are regulated by eIF3jb in cancer contexts?

  • Does manipulation of eIF3jb levels affect cancer cell growth, as demonstrated for eIF3b?

  • Is eIF3jb involved in stress adaptation mechanisms in cancer cells?

This research direction is supported by findings that eIF3b expression correlates with tumor grade, stage, and survival in human bladder and prostate cancers , suggesting that related factors like eIF3jb may play important roles in cancer biology.

What methodological approaches can be used with eIF3jb antibodies to investigate its role in neurodevelopmental processes in zebrafish?

Given eIF3jb's expression in midbrain and other neural structures , investigating its role in neurodevelopment requires specialized approaches:

• Developmental analysis techniques:

  • Whole-mount immunohistochemistry at different developmental stages

  • Co-staining with neural markers (HuC/D, acetylated tubulin)

  • Time-lapse imaging of eIF3jb dynamics during neural development

  • Electrophysiological assessments following eIF3jb manipulation

• Neural-specific methodologies:

  • Brain slice immunohistochemistry for detailed localization

  • Synaptoneurosome preparation and eIF3jb immunoblotting

  • Local translation assays in axons and dendrites

  • Behavioral testing following eIF3jb manipulation

• Experimental design considerations:

  • Use neuron-specific promoters for conditional eIF3jb manipulation

  • Correlate eIF3jb expression with neurogenesis markers

  • Examine eIF3jb in response to neural activity and neuronal plasticity

  • Investigate eIF3jb in models of neurological disorders

• Advanced imaging approaches:

  • Super-resolution microscopy for subcellular localization

  • FRAP (Fluorescence Recovery After Photobleaching) to assess eIF3jb mobility

  • Two-photon imaging for deep tissue visualization

  • Expansion microscopy for enhanced resolution in neural tissue

This research direction leverages eIF3jb's expression pattern in zebrafish neural tissues and the emerging understanding of translational regulation in neurodevelopment.