EEF1A1 Antibody, FITC conjugated

What is EEF1A1 and what cellular processes does it participate in?

EEF1A1, also known as EF1A, EF-Tu, or LENG7, belongs to the GTP-binding elongation factor family. It functions primarily as a mediator of protein synthesis by promoting GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during translation elongation. Beyond its canonical role in translation, EEF1A1 participates in multiple cellular processes including:

• Protection of the aminoester bond against hydrolysis during codon-anticodon matching

• Regulation of the actin cytoskeleton and cell morphology

• Mediation of cell death pathways, particularly during stress responses

• Participation in the heat shock response from transcription through translation

• Involvement in lipotoxicity mechanisms in hepatocytes

EEF1A1 is considered a housekeeping gene product required for cell proliferation and survival. During the heat shock response, EEF1A1 rapidly activates transcription of HSP70 by recruiting heat shock factor 1 (HSF1) to its promoter, then associates with elongating RNA polymerase II and stabilizes HSP70 mRNA .

What applications are EEF1A1 antibodies, FITC conjugated typically used for?

FITC-conjugated EEF1A1 antibodies are primarily utilized in fluorescence-based detection applications where direct visualization of EEF1A1 is required. The recommended applications include:

These applications allow researchers to visualize the subcellular localization of EEF1A1 and quantify its expression levels in individual cells . The FITC conjugation eliminates the need for secondary antibody incubation, reducing experimental time and potential background signal.

What are the optimal storage conditions for EEF1A1 antibody, FITC conjugated?

For maximum stability and performance, FITC-conjugated EEF1A1 antibodies should be stored according to these guidelines:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles that can compromise antibody integrity

• Protect from light exposure, as FITC is photosensitive

• Most formulations contain preservatives (e.g., 0.03% Proclin 300) and stabilizers (e.g., 50% Glycerol in PBS, pH 7.4)

• Properly stored, the antibody typically remains stable for one year after shipment

• Aliquoting is not necessary for -20°C storage according to some manufacturers, though it may be advisable for frequent users

Following these storage recommendations ensures optimal performance and extends the usable lifetime of the reagent.

How does EEF1A1 subcellular localization change during cellular stress, and how can this be effectively visualized?

EEF1A1 undergoes dynamic subcellular redistribution during cellular stress, particularly in response to lipotoxicity and heat shock. Research has demonstrated that:

Under basal conditions, EEF1A1 predominantly localizes to the endoplasmic reticulum (ER) in hepatocytes, where it participates in protein synthesis. Following exposure to high levels of saturated fatty acids like palmitate, EEF1A1 protein is modestly induced and partially redistributes from the ER to polymerized actin at the cell periphery. This redistribution precedes cell death and occurs within 6 hours of palmitate exposure .

To effectively visualize these changes:

• Perform time-course immunofluorescence experiments using FITC-conjugated EEF1A1 antibody (1:100-1:200 dilution)

• Co-stain with ER markers (e.g., calnexin or PDI) and actin cytoskeleton markers (e.g., phalloidin)

• Include counterstains for lipid droplets (Oil Red O or BODIPY) when studying lipotoxicity

• Use confocal microscopy with z-stack acquisition to accurately resolve subcellular compartments

• Implement quantitative image analysis to measure colocalization coefficients between EEF1A1 and organelle markers before and after stress induction

This approach allows for precise tracking of EEF1A1 translocation between subcellular compartments during stress responses.

What methodological considerations should be addressed when using EEF1A1 antibody, FITC conjugated in flow cytometry for stress response studies?

When implementing flow cytometry with FITC-conjugated EEF1A1 antibody for stress response studies, researchers should address several methodological considerations:

• Fixation and permeabilization protocol optimization:

  • Use 4% paraformaldehyde fixation (10-15 minutes) followed by permeabilization with 0.1% Triton X-100 or saponin-based buffers

  • For heat shock studies, immediate fixation is critical to capture transient responses

• Antibody titration:

  • Establish optimal concentration (starting with 0.80 μg per 10^6 cells) using unstressed and stressed cells

  • Include isotype control antibody (Rabbit IgG-FITC) at identical concentration

• Multiparameter panel design:

  • Include markers for cell viability (e.g., 7-AAD) to exclude dead cells

  • Consider dual staining for HSP70 or phospho-eIF2α to correlate with EEF1A1 levels and stress response activation

  • When studying lipotoxicity, incorporate BODIPY for lipid accumulation assessment

• Instrument settings:

  • Optimize PMT voltage for FITC channel (excitation 493 nm, emission 522 nm)

  • Implement compensation when using multiple fluorophores

• Controls and normalization:

  • Include unstressed, stressed, and recovery time points

  • Normalize mean fluorescence intensity to unstressed baseline

  • Consider using median fluorescence intensity for non-normally distributed data

These methodological considerations ensure robust and reproducible flow cytometry data when investigating EEF1A1 dynamics during cellular stress responses.

How should researchers design experiments to differentiate between EEF1A1's roles in protein synthesis versus cytoskeletal regulation?

Differentiating between EEF1A1's distinct functions requires careful experimental design that isolates each role. A comprehensive approach includes:

• Pharmacological separation:

  • Utilize didemnin B to specifically inhibit EEF1A1's peptide elongation activity without affecting its cytoskeletal functions

  • Compare with cytochalasin D treatment, which disrupts actin polymerization

  • Measure protein synthesis rates using puromycin incorporation or 35S-methionine labeling alongside cytoskeletal organization markers

• Domain-specific mutant expression:

  • Generate and express EEF1A1 constructs with point mutations in either:

    • GTP-binding domain (affecting translation function)

    • Actin-binding domain (affecting cytoskeletal function)

  • Perform rescue experiments in cells with reduced endogenous EEF1A1

• Cellular localization analysis:

  • Implement FITC-conjugated EEF1A1 antibody (1:100 dilution) for immunofluorescence

  • Co-stain for ribosomal markers (translation sites) and filamentous actin

  • Quantify colocalization coefficients under normal and stress conditions

  • Perform subcellular fractionation followed by Western blotting to confirm microscopy observations

• Functional readouts:

  • Measure translation efficiency (polysome profiling, ribosome footprinting)

  • Assess actin dynamics (FRAP of fluorescently-tagged actin)

  • Evaluate cell morphology changes (measure cell elongation, area, and perimeter)

This multifaceted approach allows for discrimination between EEF1A1's translational and cytoskeletal functions while revealing potential mechanistic connections between these activities.

What experimental controls should be included when studying EEF1A1's involvement in the heat shock response using immunofluorescence?

When investigating EEF1A1's role in the heat shock response using FITC-conjugated EEF1A1 antibody for immunofluorescence, the following comprehensive controls should be implemented:

• Antibody specificity controls:

  • Isotype control (Rabbit IgG-FITC) at equivalent concentration

  • EEF1A1 knockdown cells (using validated shRNA or siRNA)

  • Peptide competition assay using immunizing peptide

  • Include EEF1A2 staining (tissue-specific isoform) for comparison

• Treatment controls:

  • Untreated cells (basal condition)

  • Time-course heat shock treatment (42°C for 0, 15, 30, 60 minutes)

  • Recovery time points (return to 37°C for 1, 3, 6 hours)

  • Alternative stress inducers (e.g., sodium arsenite, thapsigargin) to compare stress-specific responses

• Co-staining controls:

  • HSF1 localization (to identify stress granules and confirm stress response)

  • RNA polymerase II phosphorylation status (marker of transcriptional activity)

  • HSP70 induction (downstream readout of effective heat shock response)

  • Nuclei counterstain (DAPI)

• Technical controls:

  • Secondary-only control (when using indirect immunofluorescence)

  • Autofluorescence control (unstained cells)

  • Fixed exposure parameters across all conditions

  • Z-stack acquisition to ensure complete cellular visualization

These controls ensure reliable interpretation of EEF1A1's dynamic behavior during heat shock response and facilitate distinction between specific biological effects and technical artifacts.

What are common issues encountered when using EEF1A1 antibody, FITC conjugated, and how can they be resolved?

Researchers frequently encounter several challenges when working with FITC-conjugated EEF1A1 antibodies. Here are common issues and their methodological solutions:

• High background signal:

  • Cause: Insufficient blocking, non-specific binding, or autofluorescence

  • Resolution:

    • Increase blocking time (1-2 hours) with 5% BSA or 10% normal serum

    • Add 0.1-0.3% Triton X-100 to antibody diluent to reduce non-specific binding

    • Include 0.1% Tween-20 in wash buffers and increase washing frequency

    • Perform additional washing steps (5-6 times, 5 minutes each)

    • Include 10mM NH4Cl treatment post-fixation to reduce autofluorescence

• Weak or absent signal:

  • Cause: Antibody degradation, insufficient permeabilization, epitope masking

  • Resolution:

    • Verify storage conditions and minimize light exposure

    • Optimize fixation (4% PFA, 10 minutes) and permeabilization (0.2% Triton X-100, 10 minutes)

    • Try antigen retrieval methods if working with fixed tissues

    • Increase antibody concentration (test 1:50 to 1:100 dilutions)

    • Extend incubation time (overnight at 4°C)

• Inconsistent staining patterns:

  • Cause: Sample preparation variability, cell cycle differences, cell-to-cell variability

  • Resolution:

    • Standardize cell culture conditions (density, passage number)

    • Synchronize cells for cell cycle-dependent studies

    • Include co-staining with cell cycle markers

    • Increase sample size and quantify multiple fields of view

• Photobleaching during imaging:

  • Cause: FITC sensitivity to prolonged light exposure

  • Resolution:

    • Add anti-fade reagents to mounting medium

    • Minimize exposure time during image acquisition

    • Capture FITC channel first in multi-channel imaging

    • Consider using alternative antibodies with more photostable fluorophores

Implementing these troubleshooting approaches will significantly improve staining quality and reproducibility when using FITC-conjugated EEF1A1 antibodies.

How can researchers appropriately analyze and interpret EEF1A1 expression changes in lipotoxicity models?

Analyzing EEF1A1 expression changes in lipotoxicity models requires careful consideration of multiple parameters and analytical approaches:

• Quantitative analysis methodologies:

  • Flow cytometry analysis:

    • Report median fluorescence intensity rather than mean (less affected by outliers)

    • Perform histogram overlay analysis to detect population shifts

    • Use statistical tests appropriate for flow data (Kolmogorov-Smirnov test)

  • Immunofluorescence quantification:

    • Conduct subcellular intensity distribution analysis (nuclear vs. cytoplasmic vs. peripheral)

    • Measure colocalization with ER markers and actin using Pearson's or Mander's coefficients

    • Quantify cell morphology parameters alongside intensity measurements

• Time-course considerations:

  • Analyze early (1-6h) and late (12-24h) time points separately

  • Account for the modest induction of EEF1A1 (typically 20-30% increase) at early timepoints

  • Correlate with markers of ER stress (phospho-eIF2α, GRP78) and cell death

• Lipid composition factors:

  • Compare saturated (palmitate) vs. unsaturated (oleate) fatty acid treatments

  • Consider dose-dependent effects (ranging from 400-800 μM palmitate)

  • Analyze EEF1A1 in relation to cellular lipid droplet accumulation

• Functional correlation:

  • Correlate EEF1A1 redistribution with cytoskeletal changes

  • Assess protein synthesis rates in relation to EEF1A1 levels

  • Compare with didemnin B treatment effects to distinguish translation-dependent mechanisms

• Validation approaches:

  • Confirm antibody findings with complementary techniques (Western blot, RT-qPCR)

  • Use partial knockdown models (24% reduction reported as phenotypically relevant)

  • Consider animal model validation (e.g., ob/ob mice with hepatic steatosis)

This comprehensive analytical framework ensures rigorous interpretation of EEF1A1 dynamics in lipotoxicity models, accounting for its dual roles in protein synthesis and cytoskeletal regulation.

How can EEF1A1 antibody, FITC conjugated be utilized in multiplex imaging systems to study stress response pathways?

FITC-conjugated EEF1A1 antibodies can be effectively integrated into advanced multiplex imaging systems through the following methodological approaches:

• Spectral unmixing microscopy:

  • Combine EEF1A1-FITC with spectrally adjacent fluorophores (e.g., mTFP1-tagged stress granule markers)

  • Acquire spectral signatures for each fluorophore

  • Apply linear unmixing algorithms to separate overlapping emissions

  • This approach allows for 6-8 marker visualization simultaneously

• Sequential immunofluorescence:

  • Begin with EEF1A1-FITC staining at 1:100 dilution

  • Image and record coordinates

  • Perform antibody stripping (glycine-SDS buffer, pH 2.5)

  • Re-stain with additional stress response markers

  • Computationally align and overlay images from multiple rounds

• Proximity ligation assay integration:

  • Use EEF1A1-FITC in combination with primary antibodies against potential interaction partners

  • Apply PLA probes to secondary antibodies

  • Visualize protein-protein interactions as discrete puncta

  • Particularly useful for studying EEF1A1's interactions with HSF1 and elongating RNA polymerase II

• Live-cell imaging considerations:

  • Though FITC-conjugated antibodies are primarily for fixed cells, correlative approaches can be designed

  • Pre-label cells with live-cell markers before fixation and EEF1A1-FITC staining

  • Use computational registration to align live and fixed images

• Quantitative image analysis:

  • Implement machine learning algorithms for automated detection of EEF1A1 redistribution patterns

  • Perform hierarchical clustering of cellular phenotypes

  • Quantify temporal dynamics through time-course experiments

These multiplex approaches enable simultaneous visualization of EEF1A1 with components of the heat shock response machinery and stress granule formation, providing deeper insights into the dynamics of cellular stress responses.

What are the implications of EEF1A1's role in hepatocyte lipotoxicity for potential therapeutic targeting in nonalcoholic fatty liver disease (NAFLD)?

Research on EEF1A1's involvement in hepatocyte lipotoxicity reveals several potential therapeutic avenues for NAFLD intervention:

• Targeting EEF1A1 peptide elongation activity:

  • Didemnin B (a specific inhibitor of eEF1A1 elongation activity) prevents palmitate-induced hepatocyte death by:

    • Inhibiting the recovery of protein synthesis normally associated with later phases of ER stress response

    • Preventing increased GRP78 protein production during ongoing ER stress

    • Potentially reducing the burden of protein synthesis at the ER, allowing restoration of ER homeostasis

  • Developing less toxic didemnin B derivatives or alternative EEF1A1 elongation inhibitors could provide therapeutic benefit

• Isoform-specific interventions:

  • EEF1A1 and EEF1A2 (tissue-specific isoform) differ in their ability to support heat shock response

  • EEF1A2 does not support HSR, suggesting isoform-switching could modulate stress responses

  • Therapies that alter the EEF1A1:EEF1A2 ratio might be beneficial in specific contexts

• Subcellular redistribution modulation:

  • Targeting the mechanisms controlling EEF1A1 redistribution from ER to actin cytoskeleton

  • Developing compounds that prevent this stress-induced redistribution without affecting translation

  • Cytoskeletal stabilizers could potentially prevent EEF1A1-mediated promotion of cell death

• Biomarker potential:

  • Increased liver EEF1A1 protein is observed in obese mice with severe hepatic steatosis

  • Similar findings in models of lipotoxic cardiomyopathy suggest EEF1A1 responds to lipotoxicity in vivo

  • EEF1A1 levels and subcellular distribution could serve as markers of NAFLD progression

• Combination therapy approaches:

  • Targeting EEF1A1 alongside traditional NAFLD interventions

  • Combining with ER stress reducers for synergistic effects

  • Pairing with antioxidants to address oxidative stress components

These findings suggest that EEF1A1-targeted interventions could represent a novel therapeutic approach for NAFLD, particularly for preventing progression from benign steatosis to more severe forms of the disease.