EIF4G3 Antibody, FITC conjugated

What is EIF4G3 and what is its biological significance in translation initiation?

EIF4G3 (Eukaryotic translation initiation factor 4 gamma 3) is a scaffold protein that plays a crucial role in the translation initiation process. It functions as part of the eIF4F complex that mediates mRNA recruitment to the ribosome . EIF4G3 serves as a central organizing platform for the translation initiation machinery by:

• Binding to eIF4E, the cap-binding protein that recognizes the 5' mRNA cap structure

• Interacting with eIF4A, the RNA helicase that unwinds secondary structures in the 5' untranslated region (UTR)

• Associating with the eIF3 complex, which bridges the mRNA to the 40S ribosomal subunit

• Binding to PABP (poly(A)-binding protein) to facilitate circularization of mRNA

The interaction between EIF4G3 and its binding partners is tightly regulated. For instance, hypophosphorylated EIF4EBP1 competes with EIF4G3 for binding to EIF4E, leading to translation repression. Conversely, when EIF4EBP1 is hyperphosphorylated, it dissociates from EIF4E, allowing EIF4G3 to bind and promote translation initiation . This regulatory mechanism is particularly important in cancer, where hyperactive mTOR signaling can lead to excessive phosphorylation of EIF4EBP1, decreasing its ability to inhibit eIF4E and enhancing translation of oncogenic proteins .

What are the key specifications of FITC-conjugated EIF4G3 antibodies and how do they compare to unconjugated versions?

FITC-conjugated EIF4G3 antibodies offer distinct advantages for fluorescence-based detection methods. The detailed specifications of these antibodies include:

FITC-conjugated antibodies differ fundamentally from unconjugated versions in several ways:

• Direct detection capability eliminates the need for secondary antibody steps, streamlining experimental workflows.

• FITC conjugation allows direct visualization in fluorescence microscopy and flow cytometry applications.

• Special handling requirements include protection from light to prevent photobleaching.

• While unconjugated EIF4G3 antibodies can be used for diverse applications including Western blot, IP, IHC, and IF/ICC at various dilutions (WB: 1:1000-1:6000; IP: 0.5-4.0 μg for 1.0-3.0 mg protein; IHC: 1:10-1:100; IF/ICC: 1:200-1:800) , FITC-conjugated versions are optimized for flow cytometry and immunofluorescence applications.

For researchers conducting multiparameter studies, FITC-conjugated antibodies facilitate multiplexing with other fluorophores, though careful consideration of spectral overlap is essential.

How can researchers verify the specificity of FITC-conjugated EIF4G3 antibodies?

Validating antibody specificity is crucial for ensuring reliable experimental results. A comprehensive validation strategy for FITC-conjugated EIF4G3 antibodies should include multiple complementary approaches:

Genetic Validation Methods:

• RNA Interference: Transfect cells with EIF4G3-specific siRNA or shRNA and confirm reduced signal by flow cytometry. This approach verifies that the antibody is detecting the intended target.

• CRISPR/Cas9 Knockout: Generate EIF4G3 knockout cell lines to serve as definitive negative controls. The absence of signal in these cells would strongly support antibody specificity.

• Overexpression: Create cells overexpressing epitope-tagged EIF4G3 and confirm increased signal with the FITC-conjugated antibody. This provides positive validation of target recognition.

Biochemical Validation:

• Peptide Competition Assay: Pre-incubate the antibody with excess immunizing peptide (amino acids 158-176 of human EIF4G3) before cell staining. Specific binding should be blocked, resulting in signal reduction.

• Molecular Weight Verification: While not directly applicable to flow cytometry with FITC-conjugated antibodies, parallel Western blot validation with unconjugated versions can confirm detection of the expected protein size. EIF4G3 has a calculated molecular weight of 177 kDa, though observed molecular weights of 70 kDa and/or 250 kDa have been reported .

Cross-Reactivity Assessment:

• Cell Line Panel: Test the antibody across multiple cell lines with varying EIF4G3 expression levels. The antibody has been validated in several human cell lines including HeLa, HEK-293, A431, A549, and HepG2 cells .

• Related Protein Discrimination: Assess potential cross-reactivity with other eIF4G family members (eIF4G1, eIF4G2) which share sequence homology but have distinct functions .

Controls For Flow Cytometry Experiments:

• Isotype Control: Include a rabbit IgG-FITC conjugate matching the host species and isotype of the EIF4G3 antibody.

• Unstained and Secondary-Only Controls: Essential for setting appropriate gates and determining background fluorescence levels.

• Subcellular Localization: Verify that the staining pattern is consistent with EIF4G3's expected cytoplasmic localization.

Thorough validation using these approaches ensures that experimental results accurately reflect EIF4G3 biology rather than non-specific binding or artifacts.

What cell types and experimental systems are optimal for studying EIF4G3 with FITC-conjugated antibodies?

The selection of appropriate experimental systems is critical for successful EIF4G3 studies using FITC-conjugated antibodies. Based on the validation data from multiple sources, the following systems have been confirmed to work effectively:

Validated Cell Lines:

Tissue Samples:

Human gliomas tissue has been validated for immunohistochemistry applications with EIF4G3 antibodies . For optimal results with tissue samples, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 can serve as an alternative .

Experimental Systems for Specific Research Questions:

• Translation Initiation Studies: HeLa cells are particularly well-suited for studying EIF4G3's role in translation initiation complexes due to their well-characterized translation machinery and ease of genetic manipulation.

• Stress Response Analysis: Both HeLa and HEK-293 cells respond robustly to stress inducers such as sodium arsenite, making them excellent models for studying EIF4G3's behavior during stress granule formation .

• Cancer Research Applications: HepG2 and A549 cells provide valuable models for investigating EIF4G3's role in aberrant translation in cancer contexts, particularly in relation to the mTOR signaling pathway and EIF4EBP1 phosphorylation status .

• Protein-Protein Interaction Studies: HeLa cells have been successfully used for co-immunoprecipitation experiments to study EIF4G3's interactions with other translation factors .

When establishing new experimental systems, researchers should consider:

• Endogenous expression levels of EIF4G3 and its binding partners

• Compatibility with transfection methods if genetic manipulation is required

• Growth characteristics and ease of handling

• Background autofluorescence in the FITC channel (499/515 nm)

• Fixation and permeabilization optimization for intracellular staining

What are optimal protocols for flow cytometry using FITC-conjugated EIF4G3 antibodies?

Flow cytometry with FITC-conjugated EIF4G3 antibodies requires careful optimization of sample preparation, staining, and analysis parameters to obtain reliable and reproducible results.

Sample Preparation Protocol:

• Cell Harvest and Counting:

  • Harvest adherent cells using gentle enzymatic dissociation (e.g., 0.05% trypsin-EDTA) to minimize damage

  • Count cells and prepare aliquots of 1×10^6 cells per sample

  • Wash twice with cold PBS containing 2% FBS (FACS buffer)

• Fixation and Permeabilization:

  • Since EIF4G3 is an intracellular protein, proper fixation and permeabilization are essential

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Wash twice with FACS buffer

  • Permeabilize with 0.1% Triton X-100 in PBS for 10 minutes at room temperature

  • Alternative: Use commercial permeabilization buffers optimized for intracellular staining

Staining Protocol:

• Blocking:

  • Incubate cells in blocking buffer (FACS buffer containing 5% normal serum from the same species as secondary antibody) for 30 minutes at room temperature

  • This reduces non-specific binding and background fluorescence

• Antibody Titration:

  • Determine optimal antibody concentration via titration experiment

  • Test dilutions ranging from 1:50 to 1:800 as suggested by typical IF/ICC dilutions (1:200-1:800)

  • Select concentration that provides maximum specific signal with minimal background

• Staining Procedure:

  • Incubate cells with FITC-conjugated EIF4G3 antibody at optimized dilution in FACS buffer for 45-60 minutes at room temperature in the dark

  • Wash three times with FACS buffer

  • Resuspend in 300-500 μL FACS buffer for acquisition

Critical Controls:

• Unstained Cells: For autofluorescence assessment and basic gating

• Isotype Control: Rabbit IgG-FITC conjugate at the same concentration as EIF4G3 antibody

• Positive Control: Cell line with verified high EIF4G3 expression (e.g., HeLa or HepG2)

• Negative Control: If available, EIF4G3 knockdown or knockout cells

Instrument Setup and Analysis:

• Laser and Filter Configuration:

  • Excitation: 488 nm laser line

  • Emission: 530/30 nm bandpass filter (standard FITC channel)

• Compensation:

  • If performing multicolor analysis, include single-stained controls for each fluorophore

  • Pay particular attention to PE overlap with FITC

• Gating Strategy:

  • Forward/side scatter to identify intact cells

  • Single-cell gate using FSC-H vs. FSC-A

  • Viable cell gate (if using viability dye)

  • FITC positive gate based on negative controls

• Data Reporting:

  • Report percentage of positive cells and median fluorescence intensity (MFI)

  • Include comparison to isotype control (ΔMFI)

Troubleshooting Common Issues:

• Low Signal:

  • Increase antibody concentration

  • Optimize permeabilization conditions

  • Ensure cells are adequately fixed

  • Check antibody storage conditions (avoid repeated freeze/thaw)

• High Background:

  • Increase washing steps

  • Reduce antibody concentration

  • Optimize blocking conditions

  • Ensure protection from light during handling

How can EIF4G3 antibodies be utilized to investigate translation initiation complex assembly?

FITC-conjugated EIF4G3 antibodies offer valuable tools for investigating the dynamics of translation initiation complex assembly through various experimental approaches:

Microscopy-Based Approaches:

• Co-localization Studies:

  • Perform multi-color immunofluorescence with FITC-conjugated EIF4G3 antibody and antibodies against other components of the translation machinery (eIF4E, eIF4A, eIF3 subunits)

  • Quantify co-localization using Pearson's or Mander's coefficients

  • Compare co-localization patterns under different cellular conditions (e.g., serum starvation, growth factor stimulation, stress)

• Proximity Ligation Assay (PLA):

  • Combine FITC-EIF4G3 antibody with unconjugated antibodies against potential interaction partners

  • Use secondary antibodies with oligonucleotide probes for PLA

  • Amplified fluorescent signal will occur only when proteins are in close proximity (<40 nm)

  • This approach provides greater specificity than conventional co-localization

Biochemical Approaches Combined with Flow Cytometry:

• m^7G Cap-Binding Assays:

  • Adapt the protocol described in search result #6: "Use m^7G-functionalized agarose resin to capture cap-binding complexes"

  • Elute bound proteins and analyze by flow cytometry using FITC-conjugated EIF4G3 antibody

  • Compare with other components of the eIF4F complex

  • This approach allows investigation of cap-dependent complex formation

• Co-Immunoprecipitation Studies:

  • Immunoprecipitate with antibodies against known EIF4G3 interaction partners (e.g., eIF4E)

  • Detect co-precipitated EIF4G3 using flow cytometry with the FITC-conjugated antibody

  • Compare results under different cellular conditions

  • The IP buffer composition described in can be adapted: "50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM MgCl₂, 0.5% NP-40, 10% glycerol"

Regulatory Mechanism Studies:

• mTOR Signaling Pathway Analysis:

  • Treat cells with mTOR inhibitors (e.g., rapamycin) to reduce EIF4EBP1 phosphorylation

  • Monitor changes in EIF4G3 association with EIF4E using co-IP and FITC-EIF4G3 detection

  • This approach leverages the knowledge that "hypophosphorylated [EIF4EBP1] competes with EIF4G1/EIF4G3 and strongly binds to EIF4E, leading to repress translation"

• Phosphorylation-Dependent Interactions:

  • Compare interaction patterns before and after treatment with phosphatase inhibitors

  • Examine how phosphorylation status affects complex assembly

  • According to , "the affinity between eIF4G and eIF3 is increased by insulin treatment in vivo"

Flow Cytometry Applications:

• Dual-Color Flow Cytometry:

  • Combine FITC-EIF4G3 antibody with antibodies against phosphorylated EIF4EBP1

  • Analyze correlation between EIF4EBP1 phosphorylation status and EIF4G3 associations

  • This approach allows single-cell analysis of the regulatory relationship between these factors

• Cell Cycle Analysis:

  • Combine FITC-EIF4G3 antibody with DNA content staining

  • Analyze how translation initiation complex formation varies across cell cycle phases

  • This approach connects translation regulation to cell cycle progression

The combination of these approaches provides comprehensive insights into the dynamics and regulation of translation initiation complex assembly involving EIF4G3.

What role does EIF4G3 play in stress granule formation and how can this be studied?

Stress granules are cytoplasmic RNA-protein complexes that form during cellular stress, sequestering mRNAs and translation factors. Understanding EIF4G3's role in stress granule formation provides insights into translational regulation during stress responses.

Stress Induction Protocols:

Immunofluorescence Microscopy Protocol:

• Sample Preparation:

  • Culture cells on glass coverslips

  • Apply stress treatment according to selected protocol

  • Fix immediately with 4% paraformaldehyde (10 minutes, room temperature)

  • Avoid methanol fixation which can disrupt stress granule structure

• Immunostaining:

  • Permeabilize with 0.1% Triton X-100 (10 minutes)

  • Block with 5% normal serum (1 hour)

  • Co-stain with FITC-conjugated EIF4G3 antibody and antibodies against established stress granule markers:

    • G3BP1 (stress granule nucleator)

    • TIA-1

    • PABP

    • eIF3b

  • Use spectrally distinct fluorophores that don't overlap with FITC (499/515 nm)

  • Counterstain nuclei with DAPI

• Imaging and Analysis:

  • Acquire z-stack images using confocal microscopy

  • Analyze co-localization between EIF4G3 and stress granule markers

  • Quantify number, size, and intensity of stress granules

  • Measure percentage of cells with stress granules

Flow Cytometry Approach:

• Sample Processing:

  • Apply stress treatments to cells

  • Harvest, fix, and permeabilize as described in Question 5

  • Co-stain with FITC-conjugated EIF4G3 antibody and fluorescently-labeled antibodies against stress granule markers

• Analysis Strategy:

  • Measure changes in staining pattern and intensity

  • Quantify co-expression with stress granule markers

  • Compare stressed vs. unstressed conditions

Time-Course Experiments:

Design experiments to capture the dynamics of stress granule formation and dissolution:

• Pre-stress (baseline)

• Early stress response (15-30 minutes)

• Established stress granules (60 minutes)

• Recovery phase (after stress removal)

Pharmacological Manipulations:

Combine stress treatments with inhibitors to dissect the mechanisms:

• Cycloheximide (prevents stress granule formation)

• mTOR inhibitors (affects EIF4EBP1 phosphorylation)

• Kinase inhibitors (targeting stress-activated protein kinases)

Comparative Analysis with Other EIF4G Family Members:

Based on the finding that eIF4G1 and eIF4G2 localize to stress granules while eIF4G3 may not (in Toxoplasma) , a comparative analysis would be valuable:

• Co-stain cells with antibodies against all three EIF4G family members

• Compare their localization patterns before, during, and after stress

• Investigate whether human EIF4G3 behaves differently than its Toxoplasma counterpart

This comprehensive approach would provide valuable insights into the specific role of EIF4G3 in stress granule biology and translational regulation during cellular stress.

What techniques can be used to study EIF4G3's protein-protein interactions using FITC-conjugated antibodies?

EIF4G3 functions as a scaffold protein with multiple interaction partners in the translation initiation complex. FITC-conjugated EIF4G3 antibodies can be employed in several sophisticated techniques to study these protein-protein interactions:

Flow Cytometry Protein Interaction Analysis (FCPIA):

This technique adapts traditional protein interaction methods for flow cytometry analysis:

Protocol Overview:

• Immunoprecipitate with antibodies against suspected interaction partners

• Detect co-precipitated EIF4G3 using FITC-conjugated antibody by flow cytometry

• Quantify fluorescence intensity as a measure of interaction strength

Key Advantages:

• High-throughput analysis of multiple conditions

• Quantitative measurement of interaction strengths

• Small sample requirement compared to Western blotting

Förster Resonance Energy Transfer (FRET):

FRET can detect protein interactions at molecular resolution (1-10 nm):

Implementation Strategy:

• Use FITC-EIF4G3 antibody as a donor fluorophore

• Label antibodies against interaction partners with appropriate acceptor fluorophores

• Energy transfer between fluorophores indicates close proximity of proteins

• Analyze by microscopy or flow cytometry (FRET-Flow)

Known Interaction Partners to Test:
From search results, key EIF4G3 interaction partners include:

• eIF4E1: Cap-binding protein

• eIF4A: RNA helicase

• eIF3 complex: Links 43S preinitiation complex to mRNA

• PABP: Poly(A) binding protein

Proximity Ligation Assay (PLA):

PLA offers exquisite sensitivity for detecting protein-protein interactions in situ:

Method:

• Use FITC-EIF4G3 antibody and unconjugated antibodies against potential interaction partners

• Apply secondary antibodies with attached oligonucleotides

• If proteins are in close proximity (<40 nm), oligonucleotides can be ligated

• Rolling circle amplification creates a fluorescent spot at interaction sites

• Quantify number of spots per cell as measure of interaction frequency

Applications:

• Map interactions in their native cellular context

• Visualize spatial distribution of interactions

• Detect low-abundance complexes

Bimolecular Fluorescence Complementation (BiFC) Flow Cytometry:

While not directly using FITC-EIF4G3 antibodies, this complementary approach can validate findings:

Strategy:

• Express EIF4G3 fused to one half of split fluorescent protein

• Express potential interaction partners fused to complementary half

• Interaction brings fragments together, reconstituting fluorescence

• Analyze by flow cytometry to quantify interaction frequency

• Validate results using FITC-EIF4G3 antibody staining

Quantitative Analysis of EIF4G3 Complexes:

The literature indicates that EIF4G3 forms distinct complexes:

This suggests species-specific differences in EIF4G3's interaction network that can be explored using the techniques described above.

Mapping Interaction Domains:

Flow cytometry with FITC-EIF4G3 antibodies can help map specific interaction domains:

Approach:

• Express truncated or mutated versions of EIF4G3 or its partners

• Assess interaction status by co-immunoprecipitation followed by FITC-EIF4G3 detection

• Map critical residues or domains required for interaction

For example, search result #6 mentions that eIF4G1 and eIF4G2 include the "conserved YXXXXLΦ amino acid motif that interacts with eIF4E" while eIF4G3 in Toxoplasma lacks this motif. Similar domain mapping in human EIF4G3 would provide valuable insights.

How is EIF4G3 regulated in the context of mTOR signaling, and how can this be studied?

The mTOR (mechanistic target of rapamycin) signaling pathway is a master regulator of cap-dependent translation through its effects on EIF4G3 and other translation initiation factors. Understanding this regulatory relationship provides crucial insights into translational control in normal physiology and disease states.

Regulatory Mechanism:

Based on search results #2 and #4, mTOR signaling regulates the interaction between EIF4G3 and EIF4E through EIF4EBP1 (4E-BP1) phosphorylation:

• When mTOR is inactive: EIF4EBP1 remains hypophosphorylated, strongly binds to EIF4E, and prevents EIF4G3-EIF4E interaction, thereby repressing translation .

• When mTOR is active: EIF4EBP1 becomes hyperphosphorylated, dissociates from EIF4E, allowing EIF4G3 to bind EIF4E and promote translation initiation .

• In cancer contexts: "Hyperactive mTOR signaling can lead to excessive phosphorylation of 4EBP1 decreasing its ability to inhibit eIF4E and enhancing translation of oncogenic proteins" .

Pharmacological Manipulation of mTOR Signaling:

Flow Cytometry-Based Analysis:

• Dual-Color Flow Cytometry:

  • Co-stain cells with FITC-conjugated EIF4G3 antibody and antibodies against phosphorylated EIF4EBP1

  • Analyze correlation between EIF4EBP1 phosphorylation status and EIF4G3 associations at single-cell level

  • Compare across different mTOR activity states

• Co-Immunoprecipitation with Flow Readout:

  • Immunoprecipitate EIF4E under different mTOR signaling conditions

  • Detect co-precipitated EIF4G3 using FITC-conjugated antibody by flow cytometry

  • Quantify interaction strength based on fluorescence intensity

Microscopy-Based Approaches:

• Co-localization Analysis:

  • Perform multi-color immunofluorescence with FITC-EIF4G3, EIF4E, and phospho-EIF4EBP1 antibodies

  • Quantify co-localization patterns under different mTOR signaling states

  • Analyze subcellular distribution changes upon mTOR inhibition/activation

• PLA (Proximity Ligation Assay):

  • Detect EIF4G3-EIF4E interaction events in situ

  • Quantify how interaction frequency changes with mTOR manipulation

  • Correlate with phospho-EIF4EBP1 staining in the same cells

Genetic Manipulation Approaches:

• EIF4EBP1 Mutants:

  • Express phospho-mimetic (S/T→D) or phospho-deficient (S/T→A) EIF4EBP1 mutants

  • Analyze the impact on EIF4G3-EIF4E interaction using FITC-EIF4G3 antibody

  • This approach isolates the effect of EIF4EBP1 phosphorylation from other mTOR targets

• mTOR Pathway Components:

  • Knockdown or overexpress key mTOR pathway components (RAPTOR, RICTOR, etc.)

  • Assess effects on EIF4G3 interactions and localization

  • Determine which mTOR complex (mTORC1 or mTORC2) is most important for regulation

Translational Output Assessment:

• Polysome Profiling:

  • Fractionate polysomes under different mTOR signaling conditions

  • Analyze EIF4G3 distribution across fractions using FITC-conjugated antibody

  • Correlate with translation efficiency of specific mRNAs

• Cap-Binding Assays:

  • Adapt the m^7G-resin pulldown protocol described in search result #6

  • Assess EIF4G3 recruitment to cap structures under different mTOR activity states

  • This directly tests the functional outcome of mTOR signaling on cap-dependent translation initiation

The search results provide specific experimental insights, noting that "the affinity between eIF4G and eIF3 is increased by insulin treatment in vivo" and that "the activation of mTOR complex 1 (mTORC1) is required to stabilize the interaction between these binding partners" . These observations provide a foundation for investigating the broader impact of mTOR signaling on EIF4G3's interactome.