EEFSEC Antibody

What is EEFSEC and why is it important in research?

EEFSEC (Selenocysteine-Specific Elongation Factor) is a specialized translation elongation factor that plays a critical role in the incorporation of selenocysteine into selenoproteins. Unlike the canonical translation elongation factor eEF1A, EEFSEC specifically recognizes the UGA codon in combination with a selenocysteine insertion sequence (SECIS) element to incorporate selenocysteine during protein synthesis. This process is fundamental for the production of selenoproteins, which are important for various cellular functions including antioxidant defense and redox regulation .

EEFSEC is also known as SELB in UniProt databases, with the UniProt Primary accession number P57772 for the human protein. It is encoded by the EEFSEC gene (GeneID: 60678) and has been mapped to the OMIM database under entry 607695 . The significance of EEFSEC in research stems from its unique role in selenoprotein synthesis, making it a valuable target for studies on selenium metabolism and selenoprotein function.

What are the key specifications of commercially available EEFSEC antibodies?

EEFSEC antibodies are typically polyclonal antibodies raised in rabbit hosts against human EEFSEC immunogens. These antibodies exhibit cross-reactivity with EEFSEC from multiple species including human, mouse, and rat models, making them versatile tools for comparative studies . The antibodies are generally supplied in liquid form, purified through antigen affinity chromatography, and formulated in PBS buffer (pH 7.3) containing 0.1% Sodium Azide and 50% Glycerol at concentrations around 0.35 mg/ml .

For research applications, EEFSEC antibodies have been validated for techniques such as ELISA, though optimal dilutions should be determined experimentally by individual researchers based on their specific applications and conditions . The unconjugated form of the antibody is most common, allowing researchers flexibility in choosing secondary detection methods appropriate for their experimental design.

How should EEFSEC antibodies be stored and handled for optimal results?

Proper storage and handling of EEFSEC antibodies are essential for maintaining their activity and specificity. Research-grade EEFSEC antibodies should be aliquoted upon receipt to minimize freeze-thaw cycles and stored at -20°C for long-term preservation . Repeated freeze-thaw cycles can lead to protein denaturation and loss of antibody function, so it is advisable to prepare small working aliquots.

When using the antibody for experiments, it should be thawed gradually at room temperature or on ice. For immunofluorescence applications, dilutions typically range from 1:1000 to 1:3000 in appropriate blocking solutions, though the optimal concentration should be determined empirically for each experimental setup . Working solutions can be prepared in blocking buffers containing 10% FBS to reduce non-specific binding, and incubation times may vary from 2 hours at room temperature to overnight at 4°C depending on the application .

What are the primary research applications for EEFSEC antibodies?

EEFSEC antibodies serve multiple research purposes in studying selenoprotein synthesis and EEFSEC biology. The primary applications include:

• Protein Detection: Western blotting to determine EEFSEC expression levels in different tissues or under various experimental conditions .

• Subcellular Localization Studies: Immunofluorescence microscopy to investigate the nuclear and cytoplasmic distribution of EEFSEC in different cell types, which has revealed that EEFSEC, unlike eEF1A, localizes to both the nucleus and cytoplasm .

• Protein-Protein Interaction Studies: Immunoprecipitation to identify binding partners of EEFSEC in the selenoprotein synthesis pathway.

• Functional Studies: Combining antibody-based detection with selenium supplementation experiments to assess EEFSEC's role in selenoprotein production under different conditions .

These applications have been instrumental in advancing our understanding of EEFSEC's role in selenoprotein synthesis and its potential nuclear functions.

How can researchers effectively study EEFSEC subcellular localization?

Studying EEFSEC subcellular localization requires a methodical approach combining molecular biology and imaging techniques. Based on published research, the following protocol has proven effective:

• Cell Selection: Choose appropriate cell lines for your study. McArdle 7777 rat hepatoma cells have been successfully used due to their high endogenous SBP2 expression and well-defined nuclear and cytoplasmic compartments .

• Expression System: Generate constructs expressing FLAG-tagged EEFSEC. The N-terminal FLAG tag allows for specific antibody detection without interfering with EEFSEC function. Clone the coding region into an appropriate expression vector such as pcDNA3.1 .

• Transfection Method: For McArdle cells, jetPRIME transfection has been effective. Seed cells 24 hours prior to transfection at a density of 3–4 × 10^5 per well in a 6-well plate, replace media after 24 hours, and collect cells 48 hours post-transfection .

• Immunofluorescence Protocol:

  • Seed cells on poly-L-lysine coated coverslips

  • Fix with 4% paraformaldehyde (pH 7.4) for 10 minutes

  • Block with 10% FBS, 0.2% Triton X-100 in PBS

  • Incubate with anti-FLAG antibody (1:3000) overnight at 4°C

  • Wash and add Cy3-conjugated secondary antibody (1:2500)

  • Counterstain nuclei with DAPI (1:1000 of 300 μM solution) for 10 minutes

  • Mount using Fluoromount-G and image with confocal microscopy at 63x magnification

• Quantification: Calculate the nuclear/cytoplasmic (N/C) ratio by measuring the mean fluorescence intensity in each compartment to objectively assess localization patterns .

This approach has revealed that EEFSEC consistently localizes to both the nucleus and cytoplasm, while the canonical elongation factor eEF1A is exclusively cytoplasmic, providing important insights into EEFSEC's potential nuclear functions .

What are the known nuclear localization signals in EEFSEC and how can they be investigated?

EEFSEC contains bipartite nuclear localization signals (NLS) in Domain IV that are critical for its nuclear targeting. These signals have been identified through computational prediction and experimental validation:

These results indicate that EEFSEC contains a bipartite NLS in Domain IV, with both components necessary for efficient nuclear targeting. Researchers investigating NLS in EEFSEC should consider the cooperative nature of these signals rather than focusing on individual sequences in isolation .

How does selenium status affect EEFSEC localization and function?

The relationship between selenium status and EEFSEC localization has been investigated to understand whether changes in selenoprotein synthesis affect EEFSEC distribution. The evidence indicates that:

• Experimental Approach:

  • Culture cells in selenium-supplemented (100 nM selenium) or unsupplemented media

  • Verify selenium status by measuring glutathione peroxidase 1 (GPX1) expression, which decreases dramatically in selenium-depleted conditions

  • Analyze EEFSEC localization using immunofluorescence and calculate nuclear/cytoplasmic ratios

• Research Findings:

  • No detectable GPX1 protein is observed in selenium-depleted McArdle cells, confirming reduced selenoprotein production

  • EEFSEC subcellular distribution remains unchanged regardless of selenium status

  • The nuclear localization of EEFSEC is maintained even when cells undergo significant changes in selenoprotein production

  • No changes are observed in control eEF1A localization, which remains exclusively cytoplasmic

These findings demonstrate that EEFSEC nuclear localization is independent of cellular selenoprotein production, suggesting that nuclear localization is not directly coupled to regulated selenoprotein synthesis. This indicates that EEFSEC may have nuclear functions separate from its role in selenoprotein translation .

What are the methodological considerations when overexpressing EEFSEC in cellular models?

Overexpression of EEFSEC in cellular models requires careful consideration of several methodological aspects to ensure meaningful results:

• Vector Selection and Construct Design:

  • Use expression vectors with appropriate promoters (e.g., pcDNA3.1)

  • Include epitope tags (e.g., FLAG-tag) for detection while ensuring they don't interfere with function

  • Verify construct integrity through DNA sequence analysis before transfection

• Cell Line Selection:

  • Choose cell lines with appropriate endogenous selenoprotein machinery

  • McArdle 7777 rat hepatoma cells express relatively high levels of SBP2 and show dramatic changes in selenoprotein production in response to selenium, making them suitable for EEFSEC studies

  • Consider the ease of transfection and the proportional nuclear and cytoplasmic compartments when selecting cell lines

• Transfection Optimization:

  • For transient expression, optimize transfection conditions (reagent, DNA amount, cell density)

  • For stable expression, use appropriate selection agents (e.g., G418 at 750-800 μg/mL for pcDNA3.1 vectors)

  • Verify expression by immunoblotting before proceeding with functional studies

• Functional Validation:

  • Metabolic labeling with 75Se-selenite (100 nM for 24 hours) can be used to assess the impact of EEFSEC overexpression on selenoprotein production

  • Research has shown that overexpressing EEFSEC in McArdle cells does not significantly affect selenoprotein production compared to controls, suggesting that endogenous protein is not limiting or that selenoprotein mRNAs are not accessible by the transfected pool

• Control Considerations:

  • Include appropriate controls such as empty vector and overexpression of related proteins (e.g., eEF1A) for comparison

  • Consider that experiments are conducted in the context of wild-type endogenous EEFSEC, which may affect interpretation of results

These methodological considerations are essential for designing experiments that yield reliable and interpretable data when studying EEFSEC overexpression.

What techniques are available for investigating EEFSEC's role in selenoprotein synthesis?

Multiple complementary techniques can be employed to investigate EEFSEC's role in selenoprotein synthesis:

These techniques, used in combination, provide a comprehensive toolkit for investigating EEFSEC's multifaceted role in selenoprotein synthesis and potential nuclear functions.

What are common challenges in EEFSEC antibody-based experiments and how can they be addressed?

Researchers working with EEFSEC antibodies may encounter several challenges that can be addressed through methodological adjustments:

• Specificity Issues:

  • Challenge: Cross-reactivity with other elongation factors or proteins.

  • Solution: Validate antibody specificity using appropriate controls including knockout/knockdown models or competing peptides. When using FLAG-tagged EEFSEC, consider including FLAG-tagged eEF1A as a specificity control .

• Detection Sensitivity:

  • Challenge: Low endogenous EEFSEC expression in some cell types.

  • Solution: Optimize antibody concentration through titration experiments. For immunofluorescence, prolonged incubation with primary antibody (overnight at 4°C) and signal amplification methods may improve detection .

• Background Signal:

  • Challenge: High background in immunofluorescence or immunoblotting.

  • Solution: Implement more stringent blocking protocols (10% FBS, 0.2% Triton X-100 in PBS for 1 hour at room temperature) and increase washing steps (four times with 1X PBS for immunofluorescence) .

• Antibody Stability:

  • Challenge: Loss of activity during storage.

  • Solution: Store antibody at -20°C in aliquots to avoid repeated freeze-thaw cycles. The antibody buffer (PBS, pH 7.3, containing 0.1% Sodium Azide and 50% Glycerol) helps maintain stability during storage .

• Dilution Optimization:

  • Challenge: Determining optimal antibody concentration for different applications.

  • Solution: Perform dilution series experiments for each application and cell type. While 1:3000 has been effective for immunofluorescence of FLAG-tagged EEFSEC, optimal dilutions should be determined empirically for each experimental system .

Addressing these challenges through methodological refinements will enhance the reliability and reproducibility of EEFSEC antibody-based experiments.

How can researchers validate that their EEFSEC antibody is functioning correctly?

Proper validation of EEFSEC antibodies is essential for ensuring experimental rigor. A comprehensive validation approach includes:

• Positive and Negative Controls:

  • Use cells or tissues with known EEFSEC expression levels as positive controls

  • Include EEFSEC knockout/knockdown samples as negative controls when available

  • Compare with canonical elongation factor eEF1A, which has distinct localization patterns from EEFSEC

• Size Verification:

  • Confirm detection of a protein band at the expected molecular mass (~60 kDa for human EEFSEC) by immunoblotting

  • Compare with recombinant protein standards if available

• Multiple Detection Methods:

  • Validate antibody performance across multiple techniques (Western blot, immunofluorescence, ELISA)

  • Look for consistent results across different detection methods

• Tagged Protein Comparison:

  • Express epitope-tagged EEFSEC (e.g., FLAG-EEFSEC) and detect with both anti-EEFSEC and anti-epitope antibodies

  • Concordant results indicate specific antibody recognition

• Domain Mutant Panel:

  • Test antibody against a panel of domain deletion or point mutants

  • This approach can confirm the epitope region and validate specificity

  • For example, testing against Domain IV deletion mutants would be informative if the antibody targets this region

• Cross-Species Reactivity:

  • Test the antibody against EEFSEC from different species if cross-reactivity is claimed

  • Confirm reactivity with human, mouse, and rat EEFSEC as appropriate for your research needs

• Functional Correlation:

  • Correlate antibody detection with functional assays such as selenoprotein synthesis

  • Use 75Se metabolic labeling to assess selenoprotein production in parallel with EEFSEC detection

Implementing these validation steps will ensure that experimental observations truly reflect EEFSEC biology rather than antibody artifacts.

What new insights are being gained about EEFSEC's nuclear function?

Research into EEFSEC's nuclear function is revealing intriguing aspects of this specialized elongation factor beyond its established role in selenoprotein synthesis:

• Nucleocytoplasmic Shuttling:

  • EEFSEC appears to be a nucleocytoplasmic shuttling protein based on localization studies and prediction algorithms

  • NLSMapper analysis scores (5.7 and 5.9 for the two components of the bipartite NLS) suggest this shuttling capability

  • This contrasts with canonical elongation factor eEF1A, which is exclusively cytoplasmic

• Nuclear Retention Mechanism:

  • Leptomycin B (LMB) treatment, which inhibits CRM1-dependent nuclear export, increases nuclear retention of EEFSEC

  • This indicates that EEFSEC undergoes active nuclear export rather than passive diffusion

  • The response to LMB distinguishes EEFSEC from eEF1A, which shows no response to this treatment

• Independence from Selenoprotein Synthesis:

  • Nuclear localization of EEFSEC remains constant regardless of selenium status

  • Even when selenoprotein production (e.g., GPX1) is dramatically reduced in selenium-deficient conditions, EEFSEC nuclear localization is unchanged

  • This suggests that the nuclear presence of EEFSEC is not directly coupled to its role in selenoprotein synthesis

• Domain IV Importance:

  • Domain IV of EEFSEC contains a bipartite nuclear localization signal essential for nuclear targeting

  • This domain is predicted to be flexible and prone to conformational changes

  • The bipartite nature of the NLS, with components at residues 455-487 and 513-540, suggests a complex nuclear import mechanism

These findings point to potential nuclear functions of EEFSEC that may be independent of its cytoplasmic role in selenoprotein synthesis, opening new research directions for understanding this specialized elongation factor.

How do EEFSEC domains contribute to its specialized function?

EEFSEC's unique domains contribute distinctively to its specialized function in selenoprotein synthesis and nuclear localization:

• Domain Architecture Overview:

  • EEFSEC contains multiple functional domains that distinguish it from canonical elongation factors

  • These domains contribute to its ability to specifically recognize selenocysteine insertion sequence (SECIS) elements and UGA codons

• Domain IV Functionality:

  • Contains bipartite nuclear localization signals at residues 455-487 and 513-540

  • Deletion of Domain IV results in complete nuclear exclusion

  • The sequence KKRSR (residues 536-540) was initially identified as important for nuclear localization

  • The sequence LFKKE (residues 480-484) functions as part of a bipartite NLS

  • Combined mutation of both sequences completely prevents nuclear localization

• Experimental Approaches to Study Domain Function:

  • Penta-alanine mutations of specific sequences within domains provide insight into functional regions

  • Domain deletion constructs help identify essential regions for specific functions

  • Site-directed mutagenesis targeting specific residues can pinpoint critical amino acids

  • These approaches have been successfully implemented using the QuikChange Site-Directed Mutagenesis Kit

• Domain Interaction Analysis:

  • Domains may interact with each other to facilitate proper protein function

  • Conformational changes in flexible domains like Domain IV may regulate protein activity or localization

  • Investigating these interactions requires advanced structural biology approaches combined with functional assays

Understanding the contribution of each domain to EEFSEC function provides insight into the mechanism of selenocysteine incorporation and the potential nuclear roles of this specialized elongation factor.