EEF2 Antibody

What is EEF2 and why is it important in research?

EEF2 (Eukaryotic Translation Elongation Factor 2) is a crucial protein involved in the process of protein synthesis in eukaryotic cells. It plays a pivotal role in the elongation phase of translation, facilitating the movement of the ribosome along the mRNA strand. This movement is essential for protein synthesis, making EEF2 a critical factor in fundamental cellular processes. Research has shown that EEF2 is highly expressed in excitatory neurons in the brain, with significantly higher expression in neuronal cells compared to glial cells . Its importance extends beyond basic translation, as it has been implicated in synaptic plasticity and behavior, particularly in prefrontal cortex function .

What types of EEF2 antibodies are available for research applications?

Researchers can choose from several types of EEF2 antibodies:

The choice depends on the experimental system, target species, and specific application requirements .

What dilutions should be used for different experimental applications of EEF2 antibodies?

Optimal dilutions vary by application and specific antibody. Based on technical data for antibody 67550-1-Ig:

It's critical to note that these ranges are starting points and should be optimized for each experimental system. As the documentation suggests, "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" . For other antibodies, such as the anti-eEF2 from Kerafast, a dilution of 1:10,000 is recommended for Western blotting .

How should EEF2 antibodies be stored to maintain optimal activity?

Storage conditions vary slightly between antibodies:

• Proteintech's 67550-1-Ig: Store at -20°C in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3). The antibody is stable for one year after shipment. Aliquoting is unnecessary for -20°C storage .

• Kerafast's anti-eEF2: Store at 4°C. The antibody is shipped as lyophilized whole serum and should be reconstituted in 25μL water .

• Sigma-Aldrich's ZooMAb: Specific storage conditions not mentioned in the provided information, but monoclonal antibodies typically require refrigeration .

Proper storage is essential for maintaining antibody specificity and sensitivity over time.

How does EEF2 expression vary across different cell types and brain regions?

Research has revealed significant variation in EEF2 expression across neural tissues. Immunofluorescence studies demonstrate that EEF2 is widely distributed in different brain regions but with highest expression in excitatory neurons. Colocalization analyses show that EEF2 has significantly higher colocalization with CaMKIIα (a marker of excitatory neurons) than with GAD65/67 (markers of GABAergic inhibitory neurons) . This suggests a preferential localization to excitatory neurons compared to inhibitory neurons.

Additionally, EEF2 shows differential expression across brain regions. Studies with heterozygous EEF2 knockout mice have shown that while EEF2 mRNA levels were significantly reduced in multiple brain regions (cortex, prefrontal cortex, hippocampus, and striatum), protein level reductions were most pronounced (~30%) in the prefrontal cortex, with only a reduction trend in the hippocampus and no significant changes in other regions .

What are the effects of reduced EEF2 levels on synaptic function and protein synthesis?

Reduced EEF2 levels have region-specific effects on protein synthesis and synaptic function. Research with EEF2 heterozygous knockout mice has demonstrated:

• De novo global protein synthesis was significantly reduced in the prefrontal cortex but not in other cortical regions or the hippocampus .

• Using azidohomoalanine (AHA) labeling, newly synthesized proteins were markedly decreased in the medial prefrontal cortex, primarily in CaMKIIα-positive excitatory neurons .

• Proteome analysis revealed downregulation of proteins related to mitochondrial translation, actin binding, and cell adhesion in EEF2-reduced mice .

• Synaptically, EEF2 reduction led to decreased spine density and reduced synaptic levels of GluA2 (an AMPA receptor subunit) in the prefrontal cortex .

• The synthesis of specific proteins was affected, with a remarkable decline in newly synthesized GluA2 protein in EEF2 knockdown neurons .

These findings suggest that EEF2 plays a critical role in maintaining excitatory synaptic transmission, particularly in the prefrontal cortex, by regulating the synthesis of key synaptic proteins.

What experimental controls should be included when validating the specificity of EEF2 antibodies?

When validating EEF2 antibody specificity, several controls should be implemented:

• Knockout/Knockdown Validation: Use tissue or cells with genetic deletion or knockdown of EEF2. The search results indicate that researchers verified antibody specificity using appropriate controls (Fig EV1E mentioned in result 4) .

• Multiple Antibody Comparison: Use different antibodies targeting distinct epitopes of EEF2 to confirm consistent detection patterns.

• Blocking Peptide Controls: Pre-incubate the antibody with the immunizing peptide to demonstrate specific signal reduction.

• Cross-reactivity Testing: Test the antibody against related proteins to ensure specificity. For example, the Kerafast antibody specifically does not recognize human EEF2 .

• Western Blot Molecular Weight Verification: Confirm detection at the expected molecular weight (approximately 95-100 kDa for EEF2, as reported in the search results) .

• Multi-species Validation: If the antibody claims cross-reactivity with multiple species, verify signal in each species under identical conditions.

How can EEF2 antibodies be used to study the role of EEF2 in neuronal function and disease models?

EEF2 antibodies can be utilized in multiple experimental approaches to investigate neuronal function and disease models:

• Immunohistochemistry/Immunofluorescence:

  • Map EEF2 distribution across different brain regions and cell types

  • Compare expression patterns between normal and pathological conditions

  • Analyze colocalization with neuronal markers (as done with CaMKIIα and GAD65/67)

• Western Blotting:

  • Quantify EEF2 protein levels in different brain regions

  • Monitor changes in EEF2 expression or phosphorylation in disease models

  • Analyze EEF2 levels in synaptosomal fractions to study synaptic localization

• Co-immunoprecipitation:

  • Identify EEF2-interacting proteins in neuronal cells

  • Study how these interactions change under different conditions

• Combined with Protein Synthesis Assays:

  • Use AHA labeling to measure global protein synthesis rates in conjunction with EEF2 immunostaining

  • Correlate EEF2 levels with protein synthesis rates in specific neuronal populations

• Manipulating EEF2 Levels:

  • Create knockdown or knockout models (as described in search result 4)

  • Use rescue experiments with wild-type or mutant EEF2 to establish causality

These approaches have revealed that EEF2 plays a critical role in promoting prefrontal AMPAR-mediated synaptic transmission underlying social novelty behavior, demonstrating the utility of EEF2 antibodies in understanding complex neuronal functions .

What are the optimal antigen retrieval methods for EEF2 immunohistochemistry?

For optimal antigen retrieval in EEF2 immunohistochemistry, the technical data suggests specific buffer conditions. According to the Proteintech antibody information, the recommended approach is:

• Primary suggestion: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

As noted in the documentation: "suggested antigen retrieval with TE buffer pH 9.0; (*) Alternatively, antigen retrieval may be performed with citrate buffer pH 6.0" .

The choice between these methods may depend on specific tissue types and fixation conditions. For instance, optimization might be particularly important when working with human cancer tissues, as the Proteintech antibody has been successfully used for IHC in human breast cancer and colon cancer tissues .

How can researchers optimize Western blot protocols for detecting EEF2?

Optimizing Western blot protocols for EEF2 detection requires attention to several factors:

• Sample Preparation:

  • Use appropriate lysis buffers that effectively extract EEF2 (95-100 kDa protein)

  • Include protease inhibitors to prevent degradation

  • Consider phosphatase inhibitors if studying EEF2 phosphorylation states

• Gel Electrophoresis:

  • Use 8-10% SDS-PAGE gels for optimal resolution of the 95-100 kDa EEF2 protein

  • Load appropriate protein amounts (typically 10-30 μg of total protein)

• Transfer Conditions:

  • Opt for wet transfer methods for large proteins like EEF2

  • Transfer at lower voltage for longer periods to ensure complete transfer

• Antibody Dilution:

  • Start with the recommended dilution ranges (1:5000-1:50000 for Proteintech's antibody)

  • Perform dilution series to determine optimal concentration for specific samples

• Detection System:

  • Choose appropriate secondary antibodies based on the host species (mouse for Proteintech, rabbit for others)

  • Consider enhanced chemiluminescence (ECL) systems for sensitive detection

• Validation Controls:

  • Include positive controls from cells known to express EEF2 (e.g., HeLa, HepG2, Jurkat cells)

  • Consider using recombinant EEF2 as a standard

The broad dilution range (1:5000-1:50000) suggested for the Proteintech antibody indicates high sensitivity, but emphasizes the importance of optimization for each experimental system .

What techniques can be used to study EEF2 phosphorylation and its effects on protein synthesis?

EEF2 phosphorylation is a key regulatory mechanism affecting its function in protein synthesis. Several techniques can be employed to study this process:

• Phospho-specific Antibodies:

  • Use antibodies specifically recognizing phosphorylated EEF2 (typically at Thr56)

  • Compare total EEF2 (using antibodies from the search results) with phospho-EEF2 levels

• Pharmacological Manipulation:

  • Use eEF2 kinase inhibitors to reduce phosphorylation

  • Apply conditions that activate eEF2 kinase (e.g., NMDA receptor activation in neurons)

• Protein Synthesis Assays:

  • Surface sensing of translation (SUnSET) method to measure global protein synthesis

  • AHA labeling, as mentioned in the research findings

  • Puromycin incorporation assays

• Genetic Approaches:

  • Express phosphomimetic (T56D) or phosphodeficient (T56A) EEF2 mutants

  • Compare effects on protein synthesis and cellular functions

• Mass Spectrometry:

  • Quantify phosphorylation at specific sites

  • Identify novel phosphorylation sites

• Combined Approaches:

  • Correlate phosphorylation status with protein synthesis rates in the same samples

  • Study how manipulations of EEF2 phosphorylation affect synthesis of specific proteins

These methods have revealed that EEF2 phosphorylation generally inhibits its activity, thereby reducing protein synthesis, which can have important implications for synaptic plasticity and neuronal function .

How can researchers quantify EEF2 antibody binding affinity and specificity?

Researchers can employ several techniques to quantify EEF2 antibody binding characteristics:

• Affinity Binding Assays:

  • Surface Plasmon Resonance (SPR) to determine binding kinetics

  • Bio-Layer Interferometry (BLI) for real-time measurement of binding constants

  • Enzyme-Linked Immunosorbent Assay (ELISA) for comparative affinity assessment

From the search results, we see that Sigma-Aldrich's ZooMAb was evaluated using an affinity binding assay, which determined that "a representative lot of this antibody bound EF-2/EEF2 peptide with a KD of 4.0 x 10-6 in an affinity binding assay" .

• Competitive Binding Assays:

  • Dose-dependent inhibition of antibody binding by the immunizing peptide

  • IC50 determination for quantitative comparison between antibodies

• Cross-reactivity Assessment:

  • Test binding to related proteins or EEF2 from different species

  • Evaluate binding to truncated variants or specific domains

• Epitope Mapping:

  • Peptide arrays to identify the precise binding region

  • Hydrogen-deuterium exchange mass spectrometry for conformational epitopes

• Immunoprecipitation Efficiency:

  • Quantify the percentage of target protein pulled down from a lysate

  • Compare recovery across different antibodies targeting the same protein

These quantitative approaches enable researchers to select the most appropriate antibody for their specific application and experimental system, ensuring optimal results and data reliability.

What are common issues in Western blot detection of EEF2 and how can they be resolved?

Western blot detection of EEF2 may encounter several challenges:

• High Background:

  • Issue: Non-specific binding creating background noise

  • Solution: Increase blocking time/concentration, optimize antibody dilution (starting with 1:5000 as recommended) , use more stringent washing procedures

• Weak or No Signal:

  • Issue: Insufficient antibody binding or low EEF2 expression

  • Solution: Decrease antibody dilution, increase protein loading, enhance detection system sensitivity, verify sample preparation methods

• Multiple Bands:

  • Issue: Non-specific binding or EEF2 degradation

  • Solution: Use fresher samples, add additional protease inhibitors, optimize antibody concentration, verify expected molecular weight (100 kDa as observed)

• Inconsistent Results:

  • Issue: Variable EEF2 expression or detection conditions

  • Solution: Standardize protein loading with housekeeping controls, maintain consistent experimental conditions, optimize antibody dilution for each experimental system

• Species Cross-Reactivity Issues:

  • Issue: Antibody may not recognize EEF2 from all species equally

  • Solution: Verify antibody reactivity for your species of interest (search results show reactivity with human, mouse, rat, and pig for the Proteintech antibody)

The broad dilution range recommended for Western blotting (1:5000-1:50000) indicates that careful optimization is essential for each experimental system.

How should researchers address inconsistent immunostaining patterns with EEF2 antibodies?

When facing inconsistent immunostaining patterns with EEF2 antibodies, researchers should consider:

• Fixation Protocol Optimization:

  • Issue: Overfixation or underfixation affecting epitope accessibility

  • Solution: Test different fixation times, temperatures, and fixative concentrations

• Antigen Retrieval Adjustment:

  • Issue: Insufficient epitope exposure

  • Solution: Compare TE buffer pH 9.0 with citrate buffer pH 6.0 as suggested in the technical data , or test alternative methods like pressure cooking or different retrieval durations

• Antibody Concentration Titration:

  • Issue: Sub-optimal antibody concentration

  • Solution: Perform dilution series within the recommended range (1:500-1:2000 for IHC, 1:400-1:1600 for IF/ICC)

• Blocking Optimization:

  • Issue: Insufficient blocking leading to non-specific binding

  • Solution: Test different blocking agents (BSA, normal serum, commercial blockers)

• Tissue-Specific Considerations:

  • Issue: Different tissue types may require different protocols

  • Solution: Modify protocols based on tissue characteristics, considering that EEF2 antibodies have been validated in specific tissues (e.g., human breast cancer tissue, human colon cancer tissue)

• Positive Controls:

  • Issue: Difficulty interpreting staining patterns

  • Solution: Include tissues known to express EEF2 at high levels, such as HepG2 cells, which have been validated for positive IF/ICC detection

• Secondary Antibody Matching:

  • Issue: Sub-optimal secondary antibody performance

  • Solution: Ensure appropriate species reactivity and optimize concentration

Consistent application of these optimization strategies will help overcome variability in immunostaining results.

What factors affect EEF2 antibody performance in different experimental systems?

Multiple factors can influence EEF2 antibody performance across experimental systems:

• Epitope Accessibility:

  • The accessibility of the antibody's target epitope may vary across experimental conditions

  • The Sigma-Aldrich ZooMAb targets an epitope within 19 amino acids from the C-terminal region , which may be differently exposed in various applications

• Protein Conformation:

  • Native vs. denatured conformations in different applications

  • Fixation methods may alter protein structure differently

• Species Differences:

  • Sequence homology variations between species

  • Note that the Kerafast antibody specifically does not recognize human EEF2

• Sample Preparation:

  • Lysis buffers, fixatives, and processing methods

  • Preservation of post-translational modifications

• Expression Levels:

  • EEF2 expression varies across tissues and cell types

  • Higher in excitatory neurons than inhibitory neurons or glial cells

• Antibody Format:

  • Primary antibody format (whole serum, purified IgG, recombinant)

  • The Proteintech antibody undergoes Protein G purification while the Sigma antibody is recombinantly expressed in HEK 293 cells

• Technical Variables:

  • Lot-to-lot variations in antibody production

  • Storage conditions and antibody age

Understanding these factors enables researchers to select the most appropriate antibody for their specific experimental system and optimize protocols accordingly.

How does recent research on EEF2 inform antibody selection and experimental design?

Recent research on EEF2 has revealed important insights that should guide antibody selection and experimental design:

• Cell Type Specificity: EEF2 shows differential expression patterns, with higher levels in excitatory neurons compared to inhibitory neurons or glial cells . This suggests that researchers should carefully consider cell-type-specific analyses when studying EEF2, potentially using co-staining with cell-type markers.

• Regional Variations: EEF2 function appears to be particularly important in the prefrontal cortex, with heterozygous knockout affecting protein synthesis primarily in this region . Researchers should consider region-specific analyses when studying EEF2 in the nervous system.

• Functional Significance: EEF2 has been implicated in synaptic function, particularly in AMPAR-mediated transmission . Experimental designs should consider these downstream effects when studying EEF2 manipulation.

• Target Validation: The development of genetic models (e.g., conditional knockout mice) provides valuable tools for antibody validation . Researchers should consider including such validation steps in their experimental design.

• Application-Specific Considerations: Different applications may require different antibodies, with considerations for species reactivity, clonality, and specific epitopes. The diverse antibodies described in the search results offer options for various experimental needs .

By incorporating these research insights into antibody selection and experimental design, researchers can more effectively study EEF2's role in cellular function and disease processes.

What are the most important methodological considerations when designing experiments with EEF2 antibodies?

When designing experiments with EEF2 antibodies, researchers should prioritize these methodological considerations:

• Antibody Validation: Confirm antibody specificity using knockout/knockdown controls, as demonstrated in the research findings . This is particularly important given EEF2's critical cellular functions.

• Application-Specific Optimization: Follow the recommended dilution ranges for each application (e.g., 1:5000-1:50000 for WB, 1:500-1:2000 for IHC) , but always titrate for optimal results in each experimental system.

• Species Compatibility: Ensure the selected antibody recognizes EEF2 in your species of interest. The search results show antibodies with varying species reactivity profiles .

• Proper Controls: Include positive controls (cells/tissues known to express EEF2), negative controls (knockout/knockdown samples), and technical controls (no primary antibody).

• Consideration of EEF2 Phosphorylation: EEF2 function is regulated by phosphorylation, which may affect epitope accessibility. Consider using phospho-specific antibodies alongside total EEF2 antibodies for comprehensive analysis.

• Cellular Localization: Consider subcellular fractionation or co-localization studies, given that EEF2 shows distinct patterns of expression across cell types .

• Sample Preparation: Optimize sample preparation methods, including fixation for immunostaining and lysis conditions for Western blotting, to preserve EEF2 integrity and accessibility.